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Human calcitonin receptor (hCTR) subtypes contain three or four potential Asn-linked glycosylation sites in their extracellular amino termini. The role of glycosylation in hCTR function has not been identified, but it has been suggested that inhibition of glycosylation does not affect binding or signaling. To determine the role of glycosylation in hCTR biology, we studied the effects of inhibition of glycosylation and of substitution of Asn residues that are potential glycosylation sites. Native and mutated hCTRs were studied after transient expression in monkey kidney COS-1 cells. Tunicamycin, administered as part of a treatment protocol that inhibited glycosylation of all expressed receptors, decreased salmon calcitonin (sCT) binding affinities and signaling potencies at hCTRs with three or four potential glycosylation sites. In hCTR3, which contains three potential glycosylation sites at positions 26, 78, and 83, site-specific substitution of Asn-26 by Ala had no effect on sCT binding affinity or potency, whereas substitution of Asn-78 or Asn-83 lowered sCT affinity and potency. A mutant hCTR3 in which all three Asn residues were substituted with Ala exhibited no high-affinity sCT binding and potencies of several calcitonin analogues that were more than 100-fold lower than that of native hCTR3. Our data show that glycosylation is important for high-affinity binding and potency of calcitonin analogues at hCTRs.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two proteins that are similar to human CC chemokines and a G protein-coupled receptor (KSHV-GPCR) that is constitutively active. KSHV-GPCR binds a number of human CXC and CC chemokines. We showed that interferon gamma-inducible protein-10 (IP-10), a human CXC chemokine, inhibits KSHV-GPCR signaling (Geras-Raaka et al., J. Exp. Med. 188, 405-408, 1998). Here we show that viral monocyte inflammatory protein-II (vMIP-II), one of the KSHV-encoded CC chemokines, and stromal cell-derived factor 1alpha (SDF-1alpha), a human CXC chemokine that blocks infection by human immunodeficiency virus-type 1, inhibit KSHV-GPCR signaling also. If KSHV-GPCR signaling is involved in viral pathogenesis, then these chemokines may affect the course of Kaposi's sarcoma and primary effusion lymphoma.

We have studied the role of a highly conserved tryptophan and other aromatic residues of the thyrotropin-releasing hormone (TRH) receptor (TRH-R) that are predicted by computer modeling to form a hydrophobic cluster between transmembrane helix (TM)5 and TM6. The affinity of a mutant TRH-R, in which Trp279 was substituted by alanine (W279A TRH-R), for most tested agonists was higher than that of wild-type (WT) TRH-R, whereas its affinity for inverse agonists was diminished, suggesting that W279A TRH-R is constitutively active. We found that W279A TRH-R exhibited 3.9-fold more signaling activity than WT TRH-R in the absence of agonist. This increased basal activity was inhibited by the inverse agonist midazolam, confirming that the mutant receptor is constitutively active. Computer-simulated models of the unoccupied WT TRH-R, the TRH-occupied WT TRH-R, and various TRH-R mutants predict that a hydrophobic cluster of residues, including Trp279 (TM6), Tyr282, and Phe199 (TM5), constrains the receptor in an inactive conformation. In support of this model, we found that substitution of Phe199 by alanine or of Tyr282 by alanine or phenylalanine, but not of Tyr200 (by alanine or phenylalanine), resulted in a constitutively active receptor. We propose that a hydrophobic cluster including residues in TM5 and TM6 constrains the TRH-R in an inactive conformation via interhelical interactions. Disruption of these constraints by TRH binding or by mutation leads to changes in the relative positions of TM5 and TM6 and to the formation of an active form of TRH-R.

We report on the cloning of the full-length complementary DNA for the chicken TRH receptor. Although the TRH receptor has been cloned from several mammalian species, this is the first report from another vertebrate class. The ligand binding pocket, which is situated in the transmembrane helices of the mouse and rat TRH receptors, is completely conserved in the chicken receptor. Pharmacological studies (receptor binding and signaling) employing several TRH analogs revealed that there are no significant differences between the chicken and mouse receptors. These findings show that there have been considerable evolutionary constraints on TRH receptor structure and function. Several truncated forms of the chicken TRH receptor that appear to retain a part of an intron and are truncated in the putative third intracellular loop were also cloned, but were nonfunctional. This study provides a useful tool for further studies on the roles of TRH in avian growth and TSH regulation.

A G protein-coupled receptor (GPCR) is encoded within the genome of Kaposi's sarcoma- associated herpesvirus (KSHV)/human herpesvirus 8, a virus that may be involved in the pathogenesis of Kaposi's sarcoma and primary effusion lymphomas. KSHV-GPCR exhibits constitutive signaling activity that causes oncogenic transformation. We report that human interferon (IFN)-gamma-inducible protein 10 (HuIP-10), a C-X-C chemokine, specifically inhibits signaling of KSHV-GPCR. In contrast, monokine induced by IFN-gamma (HuMig), which like HuIP-10 is an agonist of C-X-C chemokine receptor 3, does not inhibit KSHV-GPCR signaling. Moreover, HuIP-10, but not HuMig, inhibits KSHV-GPCR-induced proliferation of NIH 3T3 cells. These results show that HuIP-10 is an inverse agonist that converts KSHV-GPCR from an active to an inactive state. Thus, a human chemokine inhibits constitutive signaling and cellular proliferation that is mediated by a receptor encoded by a human disease-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, which is consistently present in tissues of patients with Kaposi's sarcoma and primary effusion lymphomas, contains a gene that encodes a G protein-coupled receptor (KSHV-GPCR). We recently showed that KSHV-GPCR exhibits constitutive signaling via activation of phosphoinositide-specific phospholipase C and stimulates cell proliferation and transformation. In this study, we determined whether normal cellular mechanisms could inhibit constitutive signaling by KSHV-GPCR and thereby KSHV-GPCR-stimulated proliferation. We show that coexpression of GPCR-specific kinases (GRKs) and activation of protein kinase C inhibit constitutive signaling by KSHV-GPCR in COS-1 monkey kidney cells and in mouse NIH 3T3 cells. Moreover, GRK-5 but not GRK-2 inhibits KSHV-GPCR-stimulated proliferation of rodent fibroblasts. These data provide evidence that cell regulatory pathways of receptor desensitization may be therapeutic targets in human diseases involving constitutively active receptors.

Small ligands generally bind within the seven transmembrane-spanning helices of G-protein-coupled receptors, but their access to the binding pocket through the closely packed loops has not been elucidated. In this work, a model of the extracellular loops of the thyrotropin-releasing hormone (TRH) receptor (TRHR) was constructed, and molecular dynamics simulations and quasi-harmonic analysis have been performed to study the static and dynamic roles of the extracellular domain. The static analysis based on curvature and electrostatic potential on the surface of TRHR suggests the formation of an initial recognition site between TRH and the surface of its receptor. These results are supported by experimental evidence. A quasi-harmonic analysis of the vibrations of the extracellular loops suggest that the low-frequency motions of the loops will aid the ligand to access its transmembrane binding pocket. We suggest that all small ligands may bind sequentially to the transmembrane pocket by first interacting with the surface binding site and then may be guided into the transmembrane binding pocket by fluctuations in the extracellular loops.

Dysregulation of G-protein-coupled receptor (GPCR) function has been shown to be associated with a growing number of human diseases. In some diseases, mutation of an endogenous GPCR causes the receptor to lose the ability to bind agonist or signal (;loss of function' mutation), whereas another mutation causes the receptor to be in an active state in the absence of agonist (;gain of function' mutation), leading to ;constitutive signaling activity'. A number of constitutively active GPCRs are tumorigenic in vitro and in animal models, and cause syndromes of hyperfunction and/or tumors in humans. The recent characterization of a constitutively active GPCR in the genome of a disease-associated, human herpesvirus provides a potential novel mechanism for viral tumorigenesis.

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is a gamma-2 herpesvirus that is implicated in the pathogenesis of Kaposi's sarcoma and of primary effusion B-cell lymphomas (PELs). KSHV infects malignant and progenitor cells of Kaposi's sarcoma and PEL, it encodes putative oncogenes and genes that may cause Kaposi's sarcoma pathogenesis by stimulating angiogenesis. The G-protein-coupled receptor encoded by an open reading frame (ORF 74) of KSHV is expressed in Kaposi's sarcoma lesions and in PEL and stimulates signalling pathways linked to cell proliferation in a constitutive (agonist-independent) way. Here we show that signalling by this KSHV G-protein-coupled receptor leads to cell transformation and tumorigenicity, and induces a switch to an angiogenic phenotype mediated by vascular endothelial growth factor, an angiogenesis and Kaposi's-spindle-cell growth factor. We find that this receptor can activate two protein kinases, JNK/SAPK and p38MAPK, by triggering signalling cascades like those induced by inflammatory cytokines that are angiogenesis activators and mitogens for Kaposi's sarcoma cells and B cells. We conclude that the KSHV G-protein-coupled receptor is a viral oncogene that can exploit cell signalling pathways to induce transformation and angiogenesis in KSHV-mediated oncogenesis.

Thyrotropin-releasing hormone (TRH), like most small ligands, appears to bind within the seven transmembrane-spanning helices (TMs) of its G protein-coupled receptor (TRH-R). A role for the extracellular loops (ECLs) of TRH-R has not been established. We substituted residues in the ECLs of TRH-R and show that Tyr-181 is important for high-affinity binding because its substitution leads to a 3700-fold lowering of the estimated affinity compared to wild-type TRH-R. Using TRH analogues, we provide evidence that there is a specific interaction between Tyr-181 in ECL-2 and the pyroGlu moiety of TRH. It was previously suggested that the pyroGlu of TRH may interact with Asn-110 in TM-3 and with Asn-289 in ECL-3; N110A and N289A TRH-Rs exhibit similar apparent affinities that are only 20-30-fold lower than wild-type TRH-R. To better understand these findings, we analyzed a computer-generated model which predicts that the ECLs form an entry channel into the TRH-R TM bundle, that Tyr-181 projects into this channel and that the pyroGlu of TRH cannot simultaneously interact with residues in the TMs and ECLs. Kinetic analysis showed that the association rate of [Ntau-methyl-His]TRH with N289A TRH-R is slower than with wild-type TRH-R and largely accounts for the lower apparent affinity; the association rate with N110A TRH-R is similar to that of wild-type TRH-R. These data are consistent with the idea that there are initial interactions between TRH and the residues of a putative entry channel of TRH-R. We suggest that a role of the ECLs in all G protein-coupled receptors for small ligands may be to initially contact the ligand and allow entry into a TM binding pocket.

The native TRH receptor (TRH-R), which is a G protein-coupled receptor that signals via the phosphoinositide transduction pathway, has been assumed to be inactive in the absence of agonist. In contrast, a mutant mouse TRH-R (C335Stop TRH-R) was shown previously to exhibit constitutive (or agonist-independent) signaling activity. In this report, we measured signaling activity of TRH-Rs using a protein kinase C-responsive reporter gene instead of formation of inositol phosphate second messenger molecules. Using this more sensitive system, we show that native mouse TRH-Rs exhibit agonist-independent signaling activity that is directly proportional to the number of receptors expressed in COS-1 cells and is inhibited by negative antagonist benzodiazepine drugs. As expected, the basal signaling activity of native TRH-Rs is lower than C335Stop TRH-Rs. Constitutive activity of native TRH-Rs is not peculiar to COS-1 cells in which receptor density is markedly elevated, because it can also be demonstrated in Madin Darby canine kidney cells stably expressing mouse TRH-Rs and GH4C1 cells endogenously expressing rat TRH-Rs. These findings support the thesis that native TRH-Rs oscillate between active and inactive states. We suggest that demonstration of constitutive activity of native receptors may depend on the sensitivity of the signaling assay employed.

The human CT receptor (hCTR), which is found as three isoforms, belongs to a small, recently described subfamily of G protein-coupled receptors (GPCRs). Several mutant GPCRs have been shown to exhibit constitutive (or agonist-independent) signaling activity and cause disease in humans, but only a few GPCRs have been identified with agonist-independent activity in the wild-type (or native) form. In the hCTR subfamily, no wild-type receptor has been shown to exhibit constitutive activity and only one, a mutated receptor for PTH/PTH-related peptide, has been found with constitutive activity to cause disease in humans. We demonstrate that two wild-type isoforms of hCTR, hCTR-1 and hCTR-2, exhibit constitutive activity by showing that they cause elevation of cAMP and induction of a cAMP-responsive gene in two cell types in culture in the absence of agonist. The identical mutation that caused the PTH/PTH-related peptide receptor to be constitutively active was made in hCTR-2 and shown to have no effect on signaling. We suggest that constitutive activity of wild-type hCTR-1 and hCTR-2 may reflect an adaptation of their signaling properties to exert their regulatory function in the absence of agonist in some cell types.

Kaposi's sarcoma-associated herpesvirus (KSHV, also known as human herpesvirus 8, or HHV 8) is a virus that is consistently present in Kaposi's sarcoma and in primary-effusion (body-cavity-based) lymphomas, malignancies that occur frequently, but not exclusively, in AIDS patients. KSHV is a gamma herpesvirus with homology to herpesvirus Saimiri and Epstein-Barr virus, both of which can transform lymphocytes. Cloning of a KSHV genome fragment revealed the presence of an open reading frame encoding a putative G-protein-coupled receptor that is homologous to a G-protein-coupled receptor encoded by herpesvirus Saimiri and to human interleukin-8 receptors. Here we show that the KSHV G-protein-coupled receptor is a bona fide signalling receptor which has constitutive (agonist-independent) activity in the phosphoinositide-inositoltrisphosphate-protein kinase C pathway. Furthermore, the KSHV G-protein-coupled receptor stimulates cellular proliferation, making it a candidate viral oncogene.

Previous mutational and computational studies of the thyrotropin-releasing hormone (TRH) receptor identified several residues in its binding pocket [see accompanying paper, Perlman et al. (1996) Biochemistry 35, 7643-7650]. On the basis of the initial model constructed with standard energy minimization techniques, we have conducted 15 mixed mode Monte Carlo/stochastic dynamics (MC-SD) simulations to allow for extended sampling of the conformational states of the ligand and the receptor in the complex. A simulated annealing protocol was adopted in which the complex was cooled from 600 to 310 K in segments of 30 ps of the MC-SD simulations for each change of 100 K. Analysis of the simulation results demonstrated that the mixed mode MC-SD protocol maintained the desired temperature in the constant temperature simulation segments. The elevated temperature and the repeating simulations allowed for adequate sampling of the torsional space of the complex with successful conservation of the general structure and good helicity of the receptor. For the analysis of the interaction between TRH and the binding pocket, TRH was divided into four groups consisting of pyroGlu, His, ProNH2, and the backbone. The pairwise interaction energies of the four separate portions of TRH with the corresponding residues in the receptor provide a physicochemical basis for the understanding of ligand-receptor complexes. The interaction of pyroGlu with Tyr106 shows a bimodal distribution that represents two populations: one with a H-bond and another without it. Asp195 was shown to compete with pyroGlu for the H-bond to Tyr106. Simulations in which Asp195 was interacting with Arg283, thus removing it from the vicinity of Tyr106, resulted in a stable H-bond to pyroGlu. In all simulations His showed a van der Waals attraction to Tyr282 and a weak electrostatic repulsion from Arg 306. The ProNH2 had a strong and frequent H-bonding interaction with Arg306. The backbone carbonyls show a frequent H-bonding interaction with the OH group of Tyr282 and strong, often multiple, interactions with Arg306. Three structures, which maintained these interactions simultaneously, were selected as candidates for ligand-receptor complexes. These show persistent interactions of TRH with Ile 109 and Ile 116 in HX 3 and with Tyr310 and Ser313 in HX 7, which will be tested to refine the structure of the ligand-receptor complex. The superposition of the three structures shows the extent of structural flexibility of the receptor and the ligand in the complex. The backbone of TRH inside the receptor is in an alpha-helical conformation, suggesting that the receptor, through its interaction with the ligand, provides the energy required for the conformational change in the ligand from an extended to the folded form.

Seven transmembrane (TM) spanning, G protein-coupled receptors (GPCRs) appear to bind large glycoprotein hormones predominantly within their extracellular domains, small nonpeptidic ligands within the TM helical bundle, and peptide ligands within the extracellular domains and TM bundle. The tripeptide thyrotropin-releasing hormone (TRH, pyroGlu-His-ProNH2) may bind entirely within the TM bundle of the TRH receptor (TRH-R). We have previously demonstrated direct binding contacts between the pyroGlu of TRH and two residues in TM helix 3 (TM-3) of TRH-R and proposed a model of the binding pocket of TRH-R [Perlman, J. H., Laakkonen, L., Osman, R., & Gershengorn, M. C. (1994) J. Biol. Chem. 269, 23383-23386]. Here, we provide evidence for two additional direct interactions between TRH and TRH-R. One interaction is between the aromatic ring of Tyr 282 of TM-6 and His of TRH. This is based on a large increase in the half-maximally effective concentration (EC50) of TRH for stimulation of inositol phosphate formation by Y282A TRH-R and a loss of selectivity of this mutant receptor for TRH analogs substituted at His. We provide evidence for another interaction between Arg 306 of TM-7 and the terminal carboxamide of TRH. Using four direct interactions as anchors, a refined model of the TRH-R binding pocket was constructed using geometry optimization through energy minimization. A novel method for modeling GPCRs based on Monte Carlo and stochastic dynamics simulations is presented in the accompanying paper [Laakkonen, L. J., Guarnieri, F., Perlman, J. H., Gershengorn, M. C., & Osman, R. (1996) Biochemistry 35, 7651-7663].

Maintenance of blood glucose by the liver is normally initiated by extracellular regulatory molecules such as glucagon and vasopressin triggering specific hepatocyte receptors to activate the cAMP or phosphoinositide signal transduction pathways, respectively. We now show that the normal ligand-receptor regulators of blood glucose in the liver can be bypassed using an adenovirus vector expressing the mouse pituitary thyrotropin releasing hormone receptor (TRHR) cDNA ectopically in rat liver in vivo. The ectopically expressed TRHR links to the phosphoinositide pathway, providing a means to regulate liver function with TRH, an extracellular ligand that does not normally affect hepatic function. Administration of TRH to these animals activates the phosphoinositide pathway, resulting in a sustained rise in blood glucose. It should be possible to use this general strategy to modulate the differentiated functions of target organs in a wide variety of pathologic states.

In Xenopus oocytes injected with total rat pituitary GH3 cell RNA, thyrotropin-releasing hormone (TRH) causes the activation of the inositol lipid transduction pathway and the induction of chloride conductance via calcium-activated channels (Oron et al., 1987, Mol. Endocrinol., 1:918-925). This response exhibits characteristic prolonged latency (Oron et al., 1988, Proc. Natl. Acad. Sci. U.S.A., 85:3820-3824; Lipinsky et al., 1993, Pflugers Arch., 425:140-149). We examined the role of agonist diffusion in the extracellular medium in the generation of latency and the determination of response amplitude. An increase in the viscosity of the medium markedly prolonged the latency and decreased the amplitude of the response. Moreover, an increase in the viscosity of the medium in the immediate vicinity of the oocyte had a major effect on both the latency and the amplitude of the response, which appeared to be a result of desensitization rather than restricted diffusion of chloride to the medium. Extrapolation to [TRH] infinity yielded a diffusion-dependent latency value of 0 and a diffusion-independent latency value of 4 seconds. In low viscosity medium, at all TRH concentrations, diffusion contributed less than 2% to the latency of the response. This implied that events distal to ligand binding are responsible for a major part of latency. Analysis of the dependence of latency and amplitude of the response on [TRH] yielded Hill coefficients markedly smaller than unity, suggesting postreceptor negative modulation of the response. Preincubation of cells with a specific inhibitor of protein kinase C, chelerythrine, increased the Hill coefficients to unity and changed the shape of the Hill plot of response amplitudes. Our results suggest that at low agonist concentrations, even in a low viscosity medium, the prolonged latency allows negative effects on both latency and amplitude by a simultaneous activation of a protein kinase C.

Consecutive challenges with thyrotropin-releasing hormone (TRH) of oocytes expressing the TRH receptor (TRH-R) resulted in a pronounced desensitization, manifested as a decrease in chloride current amplitude and an increase in response latency. Exposure to low concentrations of TRH resulted in a marked decrease in the amplitude of the subsequent response to a higher concentration of the agonist, even though the second challenge was given before the onset of the response to the first challenge (within 3 - 15 s). Cellular calcium concentration ([Ca]i) did not increase within this interval, suggesting that calcium was not involved in the desensitization process. The latency of the second response, however, was either unchanged or shortened, implying additive effects of processes initiated by the first challenge. A longer interval (30 s) between the two challenges brought about a more pronounced decrease in amplitude and a prolongation of response latency. The calcium mobilization initiated by a second challenge with a high concentration of the agonist exhibited a longer latency, a lower rate of [Ca]i increase and a lower amplitude. Stimulation of co-expressed cholinergic-muscarinic ml receptors with a low concentration of acetylcholine resulted in a pronounced desensitization of the TRH response (heterologous desensitization). Activation of protein kinase C by beta-phorbol 12-myristate, 13-acetate resulted in a dose-dependent inhibition of the response to TRH, suggesting that protein kinase C was involved in desensitization. Chelerythrine, a specific inhibitor of protein kinase C, abolished a large part of the desensitization. A mutant of the TRH-R that lacks protein kinase C consensus phosphorylation sites in the C-terminal region, exhibited desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the level of thyrotropin-releasing hormone (TRH) receptor (TRH-R) mRNA appears to involve both modulation of gene transcription and of mRNA degradation. To study regulation of TRH-R mRNA degradation and circumvent any physiological effect on transcription, we use cells stably transfected with mouse TRH-R cDNA under control of the constitutively active cytomegalovirus promoter. In stably transfected GH pituitary cells, we find that phorbol 12-myristate 13-acetate (PMA), like TRH, down-regulates TRH-R mRNA by increasing the rate of TRH-R mRNA degradation. In contrast, in stably transfected AtT-20 pituitary cells and in nonpituitary cell lines, neither TRH nor PMA decreased TRH-R mRNA levels. These findings are consistent with the idea that activation of protein kinase C leads to enhanced degradation of TRH-R mRNA in a cell-type-specific manner.

The receptor for thyrotropin-releasing hormone (TRH) is a member of the seven-transmembrane-spanning, GTP-binding protein-coupled receptor family. We showed that tyrosine at position 106 in transmembrane helix 3 of the TRH receptor directly binds the ring carbonyl of the pyroglutamyl moiety of TRH (Perlman, J. H., Thaw, C. N., Laakkonen L., Bowers, C. Y., Osman, R., and Gershengorn, M. C. (1994) J. Biol. Chem. 269, 1610-1613). We now show that asparagine at position 110 of transmembrane helix 3 directly interacts with the ring N-H of the TRH pyroglutamyl moiety. Based on these findings and evidence that two transmembrane arginines are important in binding, we developed a three-dimensional model of the TRH receptor binding pocket using molecular modeling and simulation programs. The model places the binding pocket for TRH within the transmembrane domains of the receptor and predicts that multiple hydrogen-bonding interactions are involved in binding TRH. To our knowledge, this is the first model, at an atomic level of detail, of the interaction of a peptide ligand with a GTP-binding protein-coupled receptor.

We use an adenovirus vector, AdCMVmTRHR, to express thyrotropin-releasing hormone (TRH) receptors (TRH-Rs) to determine whether the size of the hormone-responsive phosphoinositide pool in mammalian cells is directly related to receptor number. Infection of HeLa cells with increasing numbers of AdCMVmTRHR caused time-dependent graded expression of TRH-Rs. Measurement of cytoplasmic free Ca2+ in individual cells permitted quantitation of the fraction of cells responsive to TRH. Infection with 100 AdCMVmTRHR particles/cell or more led to TRH responsiveness in > or = 90% of HeLa cells. Measurement of prelabeled phosphoinositides hydrolyzed during prolonged TRH stimulation assesses the size of the TRH-responsive pool. In cells infected with AdCMVmTRHR for 24 h, the size of the TRH-responsive phosphoinositide pool increased with increasing TRH-R expression. The TRH-responsive pool also increased with time after infection as the number of TRH-Rs increased. Similar observations were made in GHY and KB cells. These data confirm our previous suggestion (Cubitt, A. B., Geras-Raaka, E., and Gershengorn, M. C. (1990) Biochem. J. 271, 331-336) that there are hormone-responsive and -unresponsive pools of cellular phosphoinositides and that the maximal size of the TRH-responsive pool is directly related to the number of TRH-Rs.

Thyrotropin-releasing hormone (TRH, pyroglutamic acid-histidine-proline-amide) binds to a seven-transmembrane-spanning, G protein-coupled receptor. We tested the hypothesis that Tyr106 of the third transmembrane helix of the TRH receptor (TRH-R) binds pyroglutamyl of TRH by mutating Tyr106 to Phe and replacing the ring carbonyl of the TRH pyroglutamyl moiety with a methylene group ([Pro1]TRH). Compared to the affinity of wild-type TRH-R for TRH, the affinities of [Phe106]TRH-R for TRH and of wild-type TRH-R for [Pro1]TRH were 100,000- and 110,000-fold lower, respectively. The affinity of [Phe106]TRH-R for [Pro1]TRH was only 16-fold lower than that for TRH, demonstrating a lack of additivity of the effects of these changes in the receptor and ligand. These data provide compelling evidence that the hydroxyl group of Tyr106 of the TRH-R binds the TRH pyroglutamyl carbonyl group. To our knowledge, this represents the highest affinity, non-covalent bond yet observed between single functional groups of a GPCR and ligand and is the first delineation of a direct binding interaction between a residue in the transmembrane core of a GPCR and a specific moiety of a peptide agonist.

1. We studied the effects of three cytoskeleton-disrupting agents, colchicine (COL), vinblastine (VIN), cytochalasins, on the functional hemispheric expression of native muscarinic and acquired thyrotropin-releasing hormone receptors TRH-Rs). Responses in oocytes of common donors, which express M3-like receptors (M3Rs), were not affected by either COL or VIN on the animal hemisphere. The functional expression of M3Rs on the vegetal hemisphere was inhibited by 50%. Cytochalasin B caused a uniform inhibition (by 31-33%) of receptor functional expression on either hemisphere. 2. Oocytes of variant donors express predominantly M1-like receptors (M1Rs) on the animal and M3Rs on the vegetal hemisphere. In these oocytes, both COL and VIN caused approximately 50% inhibition of functional expression on either hemisphere. Cytochalasin B caused more extensive, though variable inhibition on both hemispheres. Both antitubulin agents had no effect on the functional expression of the TRH-Rs on either hemisphere. Cytochalasin B, however, caused an extensive inhibition of the functional expression of this receptor (by 70-75%). 3. Induction of maturation of oocytes (7-hr incubation with progesterone) resulted in a 66% decrease in the response to TRH, reflecting mainly a decrease on the animal hemisphere. Maturation in the presence of colchicine had no further effect on the activity measured on the animal hemisphere but caused a major increase in the activity on the vegetal hemisphere. This resulted in a dramatic change in animal/vegetal activity ratio (4.8 +/- 1.5 to 0.8 +/- 0.2). 4. It appears that while antitubulin drugs affect the functional expression of the three receptors at the two hemispheres differently, disruption of the microfilaments interferes uniformly with receptor functional expression. We suggest that microfilaments may be involved in a common component of the signal transduction pathway in oocytes or in the anchoring of receptors coupled to the guaninine nucleotide-binding regulatory proteins. Moreover, progesterone-induced changes in the functional organization of the signal transduction pathway appear to be controlled to a large extent by the tubulin component of the cytoskeleton.

To dissect the cellular events responsible for the prolonged latency of the response to thyrotropin-releasing hormone (TRH) in Xenopus oocytes we interfered with different steps of the signal transduction pathway. Preincubation of oocytes with cis-vaccenic acid (a membrane-fluidizing agent) shortened the latency, suggesting a contribution of membranal processes. TRH-induced depletion of cellular calcium stores prolonged latency (up to threefold), which returned to control levels upon repletion of the stores. Injection of D-2,3-diphosphoglycerate (PGA), which inhibits inositol (1,4,5)-trisphosphate (InsP3) dephosphorylation, alone evoked a small, prolonged depolarizing current and significantly shortened the latency of the response to TRH. Injection of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inactivates guanine nucleotide-binding regulatory proteins, decreased the amplitude of the response and increased latency. Injection of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S) immediately before the challenge with TRH did not shorten the latency of the response. Decreasing the effective receptor density with chlordiazepoxide, an antagonist of the TRH receptor, resulted in an extension of latency, whereas the expression of a large number of TRH receptors by injection of RNA transcribed from cloned receptor DNA (10-100 ng/oocyte) shortened the latency to below 2 s. Our results suggest that the latency of the response to TRH reflects the activation of a late step in the signal transduction sequence, most likely the release of calcium by InsP3. We propose that this process is kinetically controlled by an early rate-limiting event, involving the activation of a guanine nucleotide-binding protein by the TRH receptor.

The thyrotropin-releasing hormone (TRH)-TRH receptor (TRHR) complex undergoes rapid transformation in cells to an acid-resistant form which appears to represent internalized agonist-receptor complex. Since residues in the carboxyl terminus of other G protein-coupled receptors appear to be involved in internalization, we studied the role of this domain in the TRHR. A mutant TRHR, C335Stop, missing the last 59 residues including 2 cysteine residues, undergoes minimal transformation to an acid-resistant form even though it binds agonist with equal affinity and activates inositol phosphate second messenger formation as effectively as wild type TRHR. Two distinct domains within the carboxyl terminus between residues 335 and 368 were shown to affect transformation equally. First, a domain between residues 360 and 367 was identified because a TRHR with codon 360 mutated to a stop codon attained a steady-state level of internalized receptor that was approximately 50% of wild type TRHR, whereas a mutant with codon 368 changed to a stop was internalized to the same extent as wild type TRHR. Second, the need for a proximal Cys residue(s), Cys-335 or Cys-337, was shown by mutating these residues to Ser or Gly. We conclude that rapid internalization of the TRHR is dependent on two dissimilar domains within the receptor carboxyl terminus.

Thyrotropin-releasing hormone, TRH (< Glu-His-Proamide), and [N tau-Me-His]TRH (MeTRH) are present as neutral and positively charged forms at physiologic pH, and it was possible that they bind to the TRH receptor (TRH-R) as charged (protonated) species. Binding affinities of TRH and MeTRH to endogenous rat TRH-Rs and to transfected wild type mouse TRH-Rs decreased below pH 7.1. Half-maximal decreases in binding occurred at the approximate pK alpha values of these ligands. Asp to Ala mutations in extracellular loop 1, TM-4, and TM-5 did not decrease binding affinity, but an Asp to Ala mutation in TM-2 caused the affinity to decrease 8-fold. The pH dependences of binding of MeTRH, however, were similar in wild type and all mutant receptors and were consistent with the protonated form of MeTRH binding less well. Thus, the binding of TRH to its receptor does not involve ionic interactions and may be a prototype for binding of neutral peptide ligands to G protein-coupled receptors.

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) down-regulates TRH receptor (TRH-R) mRNA (Fujimoto, J., Straub, R.E., and Gershengorn, M.C. (1991) Mol. Endocrinol. 5, 1527-1532), at least in part, by stimulating its degradation (Fujimoto, J., Narayanan, C.S., Benjamin, J.E., Heinflink, M., and Gershengorn, M.C. (1992) Endocrinology 130, 1879-1884). Here we show that TRH regulates RNase activity in GH3 cells and that specific mRNA sequences are needed for in vivo regulation of TRH-R mRNA by TRH. TRH affected RNase activity in a biphasic manner with rapid stimulation (by 10 min) followed by a decrease to a rate slower than in control lysates within 6 h. This time course paralleled the effects of TRH on degradation of TRH-R mRNA in vivo. The regulated RNase activity was in a polysome-free fraction of the lysates and was not specific for TRH-R RNA. A truncated form of TRH-R RNA that was missing the entire 3'-untranslated region (TRHR-R5) was more stable than full-length TRH-R RNA (TRHR-WT). In contrast to TRHR-WT mRNA, TRHR-R5 mRNA and TRHR-D9 mRNA, which was missing the 143 nucleotides 5' of the poly(A) tail, were not down-regulated by TRH in stably transfected GH3 cells as their rates of degradation were not increased. These data show that TRH regulates RNase activity in GH3 cells, that the 3'-untranslated region bestows decreased stability on TRH-R mRNA and that the 3' end of the mRNA is necessary for regulation by TRH of TRH-R mRNA degradation. We present an hypothesis that explains specific regulation of TRH-R mRNA degradation by TRH in GH3 pituitary cells.

Xenopus oocytes that express mouse thyrotropin-releasing hormone receptors (TRH-Rs) after injection if RNA transcribed from TRH-R cDNA respond to THR by a depolarizing current. This response is transduced by activation of phosphoinositide-specific phospholipase C and utilizes an as yet unidentified endogenous guanine nucleotide-binding regulatory (G) protein(s). The alpha subunit of G11 and Gq have recently been shown to couple receptors to activation of phospholipase C. To determine whether there are functional differences between these proteins, we have co-expressed the TRH-R with either alpha 11 or alpha q. alpha 11 potentiated the response to TRH (by 61 +/- 16%), while alpha q inhibited the response (by 37 +/- 9%). The changes in amplitudes were accompanied by inverse changes in response latencies. These data show that alpha 11 and alpha q differentially modulate signal transduction in Xenopus oocytes.

Benzodiazepines (BZs) interact with two classes of high affinity binding sites, equilibrium dissociation constants in the nanomolar range, a neuronal or central-type and a non-neuronal or peripheral-type. The peripheral-type binding site has been shown to be present on the outer mitochondrial membrane and appears to be involved in regulation of cholesterol transport in steroid hormone-producing endocrine cells. In rat pituitary GH3 cells, BZs bind to receptors for thyrotropin-releasing hormone (TRH) and via interaction at a different site block Ca2+ influx through voltage-sensitive channels. These, however, are low affinity interactions occurring at micromolar BZ concentrations. Here, using [3H]Ro 5-4864, we report that GH3 cells also have high affinity peripheral-type BZ binding sites. Apparent equilibrium dissociation constants of 7.8 +/- 1.7 nM and 9.3 +/- 4.5 nM for [3H]Ro 5-4864 were measured with intact cells and isolated mitochondria, respectively. As predicted from studies of these sites in other cells, the order of potencies of BZs to displace [3H]Ro 5-4864 was Ro 5-4864 greater than diazepam (DZP) much greater than clonazepam (CIZP); chlordiazepoxide (CDE) did not affect binding. Nifedipine, a dihydropyridine antagonist of Ca2+ channels that has been shown to displace BZs from peripheral-type sites in other cells, was shown to be a competitive inhibitor of [3H]Ro 5-4864 binding with a half-effective concentration in the micromolar range. Ro 5-4864, however, had no effect on Ca2+ influx or efflux in mitochondria isolated from GH3 cells. Hence, GH3 cells exhibit mitochondrial, peripheral-type BZ binding sites but the role of these putative receptors in these neuroendocrine cells, which do not produce steroid hormones, is unclear.

We showed previously that TRH down-regulates TRH receptor (TRH-R) mRNA in GH3 cells by a mechanism that appears to be mediated by protein kinase C. Here we show that vasoactive intestinal peptide (VIP) down-regulates TRH-R mRNA and present evidence that this action is mediated by protein kinase A. In GH3 cells, VIP caused a time- and concentration-dependent decrease in TRH-R mRNA. This VIP effect was simulated by 8-(4-chlorophenylthio)-cAMP, forskolin, cholera toxin and 1-methyl-3-isobutylxanthine. When cells were incubated with agents that elevate cAMP and TRH or phorbol 12-myristate 13-acetate, the decrease in TRH-R mRNA was greater than with either agent alone. When cells were pre-incubated with H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, the effects of VIP, TRH and phorbol 12-myristate 13-acetate were inhibited. We suggest that VIP, via protein kinase A, and TRH, via protein kinase C, dually regulate TRH-R mRNA.

Previous reports have provided conflicting evidence as to whether the response to TRH desensitizes. Here we show that TRH stimulation of phosphoinositide (PPI) hydrolysis, measured as inositol phosphate accumulation in the presence of LiCl, desensitizes in rat pituitary GH3 cells and in rat glioma C6 cells stably transfected with mouse pituitary TRH receptor complementary DNA. In GH3 cells, the rate of stimulation by 1000 nM TRH of PPI hydrolysis was maximal initially and then decreased by 44 +/- 13% after 20 min. In an experimental paradigm in which PPI hydrolysis was measured by adding 20 mM LiCl at different times after TRH, desensitizations caused by 3, 10, and 1000 nM TRH were 33 +/- 5%, 41 +/- 6%, and 69 +/- 2%, respectively. In transfected C6 cells, TRH-induced desensitization of 76 +/- 9% was found. In GH3 cells, 1 microM phorbol myristate acetate (PMA), an activator of protein kinase C, inhibited the initial response to TRH by 75 +/- 6% and preexposure to PMA and TRH decreased the rate of PPI hydrolysis by 98 +/- 1% after 60 min. One hundred micromolar H-7 (1-(5-isoquinolinesulfonyl)-2-methyl piperazine), an inhibitor of protein kinases, abolished the effect of PMA but did not inhibit TRH-induced desensitization. Elevation of cytoplasmic free Ca2+ by K+ depolarization increased TRH stimulation of PPI hydrolysis. We conclude that TRH stimulation of PPI hydrolysis acutely desensitizes and that this effect is not specific to pituitary cells. TRH-induced desensitization, moreover, does not appear to be mediated by protein kinase C or by elevation of cytoplasmic free Ca2+.

In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRH-Rs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/calmodulin-dependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 microM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 microM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 microM TRH. H-7 (20 microM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents.(ABSTRACT TRUNCATED AT 250 WORDS)

TRH, which does not elevate cyclic AMP, and elevation of cellular cyclic AMP decrease the density (down-regulate) of TRH receptors (TRH-Rs) on pituitary (GH3) cells. In this study we measured the effects of TRH and elevation of cyclic AMP on TRH-Rs expressed in non-pituitary cells transfected with a recently cloned mouse pituitary TRH-R complementary DNA. In stably transfected rat glioma (C6-2) cells and transiently transfected COS-1 cells TRH caused TRH-R down-regulation while elevation of cyclic AMP caused increases in TRH-R density. Hence, the effects of cyclic AMP on TRH-Rs in transfected C6-2 and COS-1 cells are different from those in GH3 cells while the effects of TRH on TRH-R are similar in all three cell types. These data show that regulation of TRH-Rs is cell type specific.

Human endothelial cell thrombin receptors were functionally expressed in Xenopus laevis oocytes by injection of RNA extracted from human umbilical vein endothelial cells. Oocytes injected with endothelial cell RNA responded to thrombin with a Ca2(+)-dependent depolarizing current whose size depended on the amount of RNA injected. In oocytes expressing thrombin receptors, thrombin caused homologous but not heterologous desensitization. Both the catalytic and anion-binding exosites of thrombin were necessary to elicit depolarizing currents. Thus, Xenopus laevis oocytes injected with mRNA from human endothelial cells express Ca2(+)-dependent thrombin receptors which share many common features with thrombin receptors on intact endothelial cells. Xenopus oocytes may, therefore, be used as a screening system in the expression cloning of the endothelial cell thrombin receptor.

Acetylcholine (ACh) and thyrotropin-releasing hormone (TRH) utilize inositol 1,4,5-trisphosphate (IP3) as a second messenger and evoke independent depolarizing membrane electrical responses accompanied by characteristic 45Ca efflux profiles in Xenopus laevis oocytes injected with GH3 pituitary cell mRNA. To determine whether this could be accounted for by mobilization of calcium from functionally separate stores, we measured simultaneously 45Ca efflux and membrane electrical responses to ACh and TRH in single oocytes. We found that depletion of ACh-sensitive calcium store did not affect the membrane electrical response to TRH and the TRH-evoked 45Ca efflux. Our data suggest that ACh and TRH mobilize calcium from distinct cellular stores in the oocyte. This is the first demonstration in a single cell of strict subcellular compartmentalization of calcium stores coupled to two different populations of cell membrane receptors that utilize the same second messenger.

Sphingosine inhibition of [3H] [N3-Me-His] TRH (MeTRH) binding, previously shown to be independent of its effects on protein kinase-C, has been further characterized in GH3 cell membranes and in a partially purified, digitonin-solubilized receptor preparation. In membranes, as in intact cells, sphingosine inhibited [3H]MeTRH binding by decreasing receptor affinity, but, in contrast to its effect in intact cells, did not affect the number of available binding sites. The inhibition of binding was linear up to 75 microM sphingosine (in the presence of 100 microM BSA at 0.1 mg membrane protein/ml), yielding an apparent Ki of 51 microM. Since GTP decreases the affinity for MeTRH binding in GH3 cell membranes, we studied interactions between GTP and sphingosine. While the effects of low concentrations of GTP gamma S and sphingosine were additive, sphingosine inhibition of MeTRH binding surpassed and was not affected by the addition of maximally inhibitory concentrations of GTP gamma S. Also, sphingosine (75 microM) did not affect the ability of a maximally effective dose of TRH to stimulate the low Km GTPase (vehicle, +35 +/- 5%; sphingosine, +32 +/- 10%); there was a 25% decrease in total GTPase activity in the presence of sphingosine. MeTRH binding to digitonin-solubilized receptors, which had properties similar to those described previously by others, including no effect of GTP on binding, was inhibited by sphingosine. In solubilized receptors, as in membranes, sphingosine caused a decrease in apparent affinity without changes in the number of binding sites. These data suggest that sphingosine interacts directly with the TRH receptor [or an associated factor(s) in the receptor complex] to decrease affinity by a mechanism that does not involve uncoupling of G-proteins.

We reported that TSH, which stimulates cAMP accumulation and proliferation of FRTL-5 thyroid cells, chronically increases the 1,2-diacylglycerol (1,2-DG) content of FRTL-5 cells. Because activation of inositol lipid hydrolysis by a phospholipase-C enzyme would generate 1,2-DG, we compared the effects of TSH on inositol lipid metabolism to TSH-induced increases in 1,2-DG content and stimulation of cAMP accumulation and cell growth. Acute stimulation of inositol lipid hydrolysis did not occur with doses of 1000 microU/ml or lower, but did occur with TSH doses of 3000 microU/ml and higher, with rates between 1-4%/h. More importantly, in cells chronically exposed to TSH, the rate of inositol lipid hydrolysis was increased only at TSH doses of 10,000 microU/ml or greater, and the maximum rate was 4-5%/h. When cells were growth arrested by TSH deprivation, there was no change in the content of inositol phosphates or polyphosphoinositides. In contrast to the high doses of TSH required to stimulate inositol lipid hydrolysis, TSH-induced elevation of 1,2-DG content and stimulation of cAMP accumulation and growth occurred at physiological TSH concentrations, with minimal effective doses in the range of 1-10 microU TSH/ml, and half-maximally effective doses between 50-200 microU TSH/ml. These data suggest that inositol lipid hydrolysis does not mediate the proliferative response to TSH in FRTL-5 cells and is not the mechanism by which increases in 1,2-DG content occur at physiological TSH concentrations.

TRH stimulation of prolactin secretion is thought to be mediated by an elevation of free cytoplasmic Ca2+. However, whether TRH-induced influx of extracellular Ca2+ is required to elevate cytoplasmic Ca2+ remains controversial. We measured cytoplasmic free Ca2+ concentration in GH3 cells with an intracellularly trapped fluorescent indicator, Quin 2. In unstimulated cells incubated in medium containing 1.5 mM Ca2+, cytoplasmic free Ca2+ concentration was 118 +/- 18 nM (mean +/- SD). TRH (1 microM) caused a rapid transient elevation of free cytoplasmic Ca2+ to a level estimated to be at least 500 nM. High extracellular K+, which induces extracellular Ca2+ influx, caused an elevation of free cytoplasmic Ca2+ which was greater and longer in duration that that caused by TRH. When cells were incubated in medium containing 3 mM EGTA, the K+ depolarization-induced increase in free cytoplasmic Ca2+ was abolished. By contrast, the TRH-induced increase was not affected by incubating cells in medium with 3 mM EGTA, or high K+, or both; incubation of cells in medium with EGTA and high K+ abolishes the electrochemical driving force for Ca2+ influx. These data demonstrate that Ca2+ influx is not required for TRH-induced elevation of free cytoplasmic Ca2+ in GH3 cells. We conclude that in GH3 cells TRH induces an elevation of free cytoplasmic Ca2+ leading to stimulated prolactin secretion by mobilizing cellular Ca2+.

TRH stimulation of prolactin release from GH3 cells is associated with loss of cellular Ca2+. Chlortetracycline (CTC), a fluorescent probe of Ca2+ in biological membranes, was previously employed to monitor indirectly changes in membrane Ca2+ in GH3 cells. Tetracaine, propranolol and trifluoperazine, agents that are known to displace Ca2+ from biological membranes, were utilized to demonstrate more rigorously that TRH affects cellular membrane Ca2+ in GH3 cells. Tetracaine (1 mM), propranolol (1 mM), and trifluoperazine (0.03 mM) inhibited basal and TRH-stimulated prolactin release, decreased cellular 45Ca2+ content and decreased cell-associated CTC fluorescence. Most importantly, these agents abolished the decrease in CTC fluorescence induced by TRH. These data suggest that tetracaine, propranolol and trifluoperazine displace membrane Ca2+ in intact GH3 cells and offer further evidence that TRH acts to mobilize cellular Ca2+ from a membrane-bound pool(s) during stimulation of GH3 cells.

Thyroxine-binding globulin biosynthesis was demonstrated in hepatocytes isolated from normal adult Rhesus monkeys. Dispersed cells were obtained by in situ liver perfusion with collagenase, hyaluronidase and EDTA. Conditions for optimum cell survival and incorporation of radioactive leucine into newly synthesized proteins were defined. Protein synthesis, and specifically thyroxine-binding globulin synthesis, were shown to continue throughout the incubation period, while cell survival remained high (75% excluded trypan blue after 6h). Incubation medium, cytosol and a particulate fraction (extracted with digitonin) were analyzed for thyroxine-binding globulin. After extensive dialysis and purification by affinity chromatography, newly synthesized thyroxine-binding globulin was identified by specific double-antibody immunoprecipitation and by immunodiffusion and immunoelectrophoresis with autoradiography. Newly synthesized thyroxine-binding globulin was present after 4 h of incubation. After 6 h, the total synthesized had increased to 150% of the 4 h value, while the fraction present in the medium and increased to 300%, indicating probable thyroxine-binding globulin secretion

The secondary and tertiary structure of human plasma thyroxine-binding globulin (TBG) was investigated by circular dichroism and fluorescence properties. The relaxation time of TBG indicated that it is a compact, symmetric molecule. It was calculated from the far ultraviolet CD spectrum that about one-half of the peptide groups are equally distributed in alpha helical and beta structures. In the near ultraviolet, the CD spectrum of TBG was modified when thyroxine was bound. TBG was stable at temperatures below 50 degrees at pH 9 and below 35 degrees at pH 10.5. Below pH 5 tryptophanyl fluorescence revealed a molecular transition which followed first order kinetics. The transition resulted in an irreversible loss of binding of the hormone. Acidification to pH 3.4 produced only a minor change in the CD spectrum, in which some of the alpha helical peptides were converted to beta structure.

TRH stimulation of rat pituitary (GH3) cells causes biphasic changes in cytoplasmic free Ca2+ concentration [( Ca2+]i) and PRL secretion. It has been proposed, based primarily on indirect evidence, that the first phase effects are mediated by inositol 1,4,5-trisphosphate, which releases Ca2+ from cellular stores, and the sustained effects are mediated by 1,2-diacylglycerol, which activates protein kinase C. To determine more directly if inositol lipid hydrolysis leading to protein kinase C activation is involved in the sustained effects of TRH, GH3 cells were depleted of phosphatidylinositol (PtdIns) by prestimulation and incubation in myo-inositol-free, Li(+)-containing medium. Cells depleted of PtdIns (to 53 +/- 3.2% of control) had unchanged PtdIns 4,5-bisphosphate content, and responded to TRH with a rapid elevation of inositol trisphosphate, and a first phase (or burst) elevation of [Ca2+]i and PRL secretion that was not different from that found in control cells. In contrast, in PtdIns-depleted cells, the prolonged generation of inositol phosphates, which are produced in equimolar amounts with 1,2-diacylglycerol, caused by TRH was virtually abolished, and the second phase (or sustained) elevation of [Ca2+]i and PRL secretion were inhibited by 50% and 40%, respectively. The inhibition of both sustained effects was reversed by adding 100 mM myo-inositol to the medium, which allowed for synthesis of PtdIns. Last, in cells in which protein kinase C was down-regulated by pretreatment with a phorbol ester, the sustained effects of TRH were inhibited also.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum thyroxine-binding globulin (TBG) was measured by radioimmunoassay. The human TBG used in this study was purified by affinity, anion-exchange, and gel filtration chromatography. The serum TBG concentration in 98 euthyroid normals was 1.48 +/- 0.46 mg/100 ml (mean +/- SD), which is one-half that previously reported using a similar method. The level in females (1.66 +/- 0.56) was significantly higher than that in males (1.37 +/- 0.37). Comparison of the serum TBG level and the maximum binding capacity of serum TBG for thyroxine (T4) yielded a molar ratio of 1:1 for T4 and TBG. The mean serum TBG in 19 patients with hepatocellular carcinoma was 2.10 +/- 1.29 mg/100 ml; however, only 2 of these patients had serum TBG levels outside the normal range.

Primary cultures from three serially transplanted mouse pituitary thyrotropic tumors, Furth 97A, 97B, and 97C, and one newly generated tumor, NIH 101A, were shown to secrete free alpha subunits and a beta subunit (TSH-beta) in addition to intact TSH. The three glycopeptides were measured in specific, heterologous radioimmunoassays. Basal secretion of alpha subunit was found to be in excess in all four thyrotropic tumor cultures, ranging from 5.7- to 8.9-fold higher than that of intact TSH and 30- to 35-fold higher than that of free TSH-beta. In cultures from 97A, TRH (10(-7) M) stimulated the secretion of TSH, alpha subunit, and TSH-beta by 175-185% and (T4, 10(-5) M) inhibited TSH to 19%, alpha subunit to 68%, and TSH-beta to 58% of control. Secretion of TSH and its subunits in cultures from 97B was not consistently affected by TRH or T4. In cultures from 97C, TSH and alpha secretion was inhibited in a dose-dependent fashion by T3 from 2.5 x10(-9) to 1.6x10(-7) M. Gel chromatography of incubation media from tumor 97A revealed elution volumes of TSH and alpha similar to those of purified rat glycopeptides and confirmed the excess secretion of free alpha subunit. In contrast, the alpha to TSH ratios in media from cultures of pituitary cells from normal and thyroidectomized rats were 1.6 and 0.8, respectively. The total production of TSH and its subunits was measured in cultures from tumor 97A and confirmed that alpha subunit was synthesized excessively. The ratio of the plasma concentrations of alpha to TSH in tumor-bearing mice was 1.6 compared to 1.3 in thyroidectomized mice, and 0.36 in thyroidectomized rats. The alpha to TSH ratios in extracts of the corresponding thyrotropic tissues (tumor or pituitary) were 2.7, 1.7, and 0.40, respectively. These data are analogous to those reported in humans with thyrotropic and certain other pituitary tumors, and support hypothesis that the subunits of glycoprotein hormones are independently synthesized.