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Cyclooxygenases metabolize arachidonate to five primary prostanoids: PGE(2), PGF(2 alpha), PGI(2), TxA(2), and PGD(2). These autacrine lipid mediators interact with specific members of a family of distinct G-protein-coupled prostanoid receptors, designated EP, FP, IP, TP, and DP, respectively. Each of these receptors has been cloned, expressed, and characterized. This family of eight prostanoid receptor complementary DNAs encodes seven transmembrane proteins which are typical of G-protein-coupled receptors and these receptors are distinguished by their ligand-binding profiles and the signal transduction pathways activated on ligand binding. Ligand-binding selectivity of these receptors is determined by both the transmembrane sequences and amino acid residues in the putative extracellular-loop regions. The selectivity of interaction between the receptors and G proteins appears to be mediated at least in part by the C-terminal tail region. Each of the EP(1), EP(3), FP, and TP receptors has alternative splice variants described that alter the coding sequence in the C-terminal intracellular tail region. The C-terminal variants modulate signal transduction, phosphorylation, and desensitization of these receptors, as well as altering agonist-independent constitutive activity.

Prostaglandin E(2) is a major renal cyclooxygenase metabolite of arachidonate and interacts with four G protein-coupled E-prostanoid receptors designated EP(1), EP(2), EP(3), and EP(4). Through these receptors, PGE(2) modulates renal hemodynamics and salt and water excretion. The intrarenal distribution and function of EP receptors have been partially characterized, and each receptor has a distinct role. EP(1) expression predominates in the collecting duct where it inhibits Na(+) absorption, contributing to natriuresis. The EP(2) receptor regulates vascular reactivity, and EP(2) receptor-knockout mice have salt-sensitive hypertension. The EP(3) receptor is also expressed in vessels as well as in the thick ascending limb and collecting duct, where it antagonizes vasopressin-stimulated salt and water transport. EP(4) mRNA is expressed in the glomerulus and collecting duct and may regulate glomerular tone and renal renin release. The capacity of PGE(2) to bidirectionally modulate vascular tone and epithelial transport via constrictor EP(1) and EP(3) receptors vs. dilator EP(2) and EP(4) receptors allows PGE(2) to serve as a buffer, preventing excessive responses to physiological perturbations.

Prostaglandin E2 (PGE2) stimulates the formation of osteoclast-like tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNC) in vitro. This effect likely results from stimulation of adenylyl cyclase, which is mediated by two PGE2 receptors, designated EP2 and EP4. We used cells from mice in which the EP2 receptor had been disrupted to test its role in the formation of TRAP + MNC. EP2 heterozygous (+/-) mice in a C57BL/6 x 129/SvEv background were bred to produce homozygous null (EP2 -/-) and wild-type (EP2 +/+) mice. PGE2, PTH, or 1,25 dihydroxyvitamin D increased TRAP+ MNC in 7-day cultures of bone marrow cells from EP2 +/+ mice. In cultures from EP2 -/- animals, responses to PGE2, PTH, and 1,25 dihydroxyvitamin D were reduced by 86%, 58%, and 50%, respectively. A selective EP4 receptor antagonist (EP4RA) further inhibited TRAP+ MNC formation in both EP2 +/+ and EP2 -/- cultures. In cocultures of spleen and calvarial osteoblastic cells, the response to PGE2 or PTH was reduced by 92% or 85% when both osteoblastic cells and spleen cells were from EP2 -/- mice, by 88% or 68% when only osteoblastic cells were from EP2 -/- mice and by 58% or 35% when only spleen cells were from EP2 -/- mice. PGE2 increased receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) messenger RNA expression in osteoblastic and bone marrow cell cultures from EP2 +/+ mice 2-fold but had little effect on cells from EP2 -/- mice. Spleen cells cultured with RANKL and macrophage colony stimulating factor produced TRAP+ MNC. PGE2 increased the number of TRAP+ MNC in spleen cell cultures from EP2 +/+ mice but not in cultures from EP2 -/- mice. EP4RA had no effect on the PGE2 response in spleen cell cultures. PGE2 decreased the expression of messenger RNA for granulocyte-macrophage colony stimulating factor in spleen cell cultures from EP2 +/+ mice but had little effect on cells from EP2 -/- mice. These data demonstrate that the prostaglandin EP2 receptor plays a role in the formation of osteoclast-like cells in vitro. A major defect in EP2 -/- mice appears to be in the capacity of osteoblastic cells to stimulate osteoclast formation. In addition, there appears to be a defect in the response of cells of the osteoclastic lineage to PGE2 in EP2 -/- mice.

Four E-prostanoid (EP) receptors, designated EP(1), EP(2), EP(3), and EP(4), mediate the cellular effects of prostaglandin E(2) (PGE(2)). The present studies pharmacologically characterize the vasopressor and vasodepressor EP receptors in wild-type mice (EP(2)(+/+) mice) and mice with targeted disruption of the EP(2) receptor (EP(2)(-/-) mice). Mean arterial pressure (MAP) was measured via a carotid artery catheter in anesthetized male mice. Intravenous infusion of PGE(2) decreased MAP in EP(2)(+/+) mice but increased MAP in EP(2)(-/-) mice. Infusion of EP(3)-selective agonists, including MB28767, SC46275, and sulprostone, increased MAP in both EP(2)(+/+) and EP(2)(-/-) mice. Pretreatment with SC46275 desensitized mice to the subsequent pressor effect of sulprostone, but the vasodepressor effect of PGE(2) in EP(2)(+/+) mice remained intact. Although PGE(2) alone increased MAP in EP(2)(-/-) mice, prior desensitization of the pressor effect with SC46275 allowed a residual vasodepressor effect of PGE(2) to be seen in the EP(2)(-/-) mice. An EP(4)-selective agonist (prostaglandin E(1)-OH) functioned also as a vasodepressor in both EP(2)(-/-) and EP(2)(+/+) mice. High levels of EP(3) receptor mRNA were detected in mouse aortas and rabbit preglomerular arterioles by nuclease protection, with lower expressions of EP(1), EP(2), and EP(4) mRNA. The findings suggest that combined vasodepressor effects of EP(2) and EP(4) receptors normally dominate, accounting for the depressor effects of PGE(2). In contrast, in EP(2)(-/-) mice, EP(4) receptor activity alone is insufficient to overcome the EP(3) vasopressor effect. These findings suggest that a balance between pressor and depressor PGE(2) receptors determines its net effect on arterial pressure and that these receptors may be important therapeutic targets.

Prostaglandins (PGs) are ubiquitous lipid mediators derived from cyclooxygenase (COX) metabolism of arachidonic acid that exert a broad range of physiologic activities including modulation of inflammation, ovulation, and arterial blood pressure. The physiologic actions of PGs are mediated in part by their interaction with specific G-protein-coupled PG receptors. Eight PG receptors have been cloned, including four for the major COX metabolite, PGE2. The physiologic roles of the PGE2 receptors have been investigated utilizing subtype-selective agonists, localization of receptor mRNA expression, and creation of mice with targeted disruption of PG receptor genes. These analyses have delineated discrete roles for the various PG receptor subtypes. Recent studies on mice lacking the PGE2 EP2 receptor have implicated the PGE2 EP2 receptor subtype in arterial dilatation and salt-sensitive hypertension, and also indicate that this receptor plays a key role in female fertility. The EP2 receptor may thus prove to be a productive target for pharmacological intervention in the treatment of hypertension and infertility.

Renal cyclooxygenase-1 and cyclooxygenase-2 actively metabolize arachidonate to metabolism five primary prostanoids: prostaglandin E2, prostaglandin F2a, prostaglandin I2, thromboxane A2, and prostaglandin D2. These lipid mediators interact with a family of distinct G-protein-coupled prostanoid receptors designated EP, FP, IP, TP, and DP, respectively, which exert important regulatory effects on renal function. The intrarenal distribution of these prostanoid receptors has been mapped and the consequences their activation are being characterized. The FP, TP, and EP1 receptors preferentially couple to increased cell Ca2+. EP2, EP4, DP, and IP receptors stimulate cyclic adenosine monophosphate, whereas the EP3 receptor preferentially couples to Gi, inhibiting cyclic adenosine monophosphate generation. EP1 and EP3 messenger RNA expression predominate in the collecting duct and thick limb, respectively, where their stimulation reduces sodium chloride and water absorption, promoting natriuresis and diuresis. Interestingly, only a mild change in renal water handling is seen in the EP3 receptor knockout mouse. Although only low levels EP2 receptor messenger RNA are detected in kidney and its precise intrarenal localization is uncertain, mice with targeted disruption of the EP2 receptor display salt-sensitive hypertension, suggesting it also plays an important role in salt excretion. In contrast, EP4 messenger RNA is readily detected in the glomerulus where it may contribute to the regulation of renin release and decrease glomerular resistance. TP receptors are also highly expressed in the glomerulus, where they may increase glomerular vascular resistance. The IP receptor messenger RNA is most highly expressed in the afferent arteriole and it may also modulate renal arterial resistance and renin release. At present there is little evidence for DP receptor expression in the kidney. Together these receptors act as physiologic buffers that protect the kidney from excessive functional changes during periods of physiologic stress. Loss of the combined effects of these receptors contributes to the side effects seen in the setting of nonsteroidal anti-inflammatory drug administration, whereas selective antagonists for these receptors may provide new therapeutic approaches in disease.

The present study examined the expression and role of the thiazolidinedione (TZD)-activated transcription factor, peroxisome proliferator-activated receptor gamma (PPARgamma), in human bladder cancers. In situ hybridization shows that PPARgamma mRNA is highly expressed in all human transitional epithelial cell cancers (TCCa's) studied (n=11). PPARgamma was also expressed in five TCCa cell lines as determined by RNase protection assays and immunoblot. Retinoid X receptor alpha (RXRalpha), a 9-cis-retinoic acid stimulated (9-cis-RA) heterodimeric partner of PPARgamma, was also co-expressed in all TCCa tissues and cell lines. Treatment of the T24 bladder cancer cells with the TZD PPARgamma agonist troglitazone, dramatically inhibited 3H-thymidine incorporation and induced cell death. Addition of the RXRalpha ligands, 9-cis-RA or LG100268, sensitized T24 bladder cancer cells to the lethal effect of troglitazone and two other PPAR- activators, ciglitazone and 15-deoxy-delta(12,14)-PGJ2 (15dPGJ(2)). Troglitazone treatment increased expression of two cyclin-dependent kinase inhibitors, p21(WAF1/CIP1) and p16(INK4), and reduced cyclin D1 expression, consistent with G1 arrest. Troglitazone also induced an endogenous PPARgamma target gene in T24 cells, adipocyte-type fatty acid binding protein (A-FABP), the expression of which correlates with bladder cancer differentiation. In situ hybridization shows that A-FABP expression is localized to normal uroepithelial cells as well as some TCCa's. Taken together, these results demonstrate that PPARgamma is expressed in human TCCa where it may play a role in regulating TCCa differentiation and survival, thereby providing a potential target for therapy of uroepithelial cancers.

The ligand binding pocket of biogenic amine G protein-coupled receptors is embedded in the membrane-spanning regions of these receptors, whereas the extracellular domains of the peptidergic receptors play a key role in the structure and function of this class of receptors. To examine the role of the extracellular sequences in prostaglandin receptor-ligand interaction, chimeras were constructed with the two G(s)-coupled E-prostanoid (EP) receptors, replacing each of the extracellular sequences of the human EP(2) receptor with the corresponding human EP(4) receptor residues. Replacement of the third extracellular loop (ECIII) yielded a receptor that binds [(3)H]prostaglandin E(2) (PGE(2); K(d) = 6.3 nM) with similar affinity as the EP(2) wild-type receptor (K(d) = 12.9 nM). Similarly, replacement of the nonconserved carboxyl-terminal portion of ECII resulted in a receptor that maintains [(3)H]PGE(2) binding (K(d) = 8.8 nM). In contrast, replacement of the amino terminus, ECI, the entire ECII region, or the residues within the highly conserved motif of the amino-terminal half of ECII yielded chimeras that displayed neither detectable [(3)H]PGE(2) binding nor receptor-evoked cAMP generation. Immunoprecipitation demonstrated that each chimera is expressed at levels near that of wild-type receptors; however, enzyme-linked immunosorbent assay revealed that inactive chimeras have reduced cell surface expression. Similarly, chimeras that exchange the multiple extracellular loop sequences N/ECI, ECII/ECIII, or all four sequences lacked detectable binding and signal transduction, and although expressed, were not detected on the cell surface. These data suggest that the extracellular sequences of the EP(2) receptor are critical determinants of receptor structure and/or function, unlike other G protein-coupled receptors that bind small molecules.

The prostaglandin E-prostanoid (EP)3 receptor signals primarily through the inhibitory G protein Gi, thereby decreasing intracellular cAMP levels. To study the signal transduction properties of the rabbit EP3 receptor, five splice variants were expressed in HEK293tsA201 cells: 72A, 74A, 77A, 80A and the novel splice variant NT, which lacks the C-terminal sequence. The ability of the EP3 receptor splice variants to modulate expression of a beta-galactosidase reporter gene under the control of a promoter containing cAMP response elements (CRE) was assessed. Each splice variant induced sulprostone-mediated increase in beta-galactosidase enzymatic activity with EC50 ranging from 0.8 nM for the NT splice variant to 3.1 nM for the 77A splice variant. Substitution of either Asp338 with Ala, or Arg329 with Ala or Glu in the 77A splice variant resulted in a loss of receptor-evoked increases in beta-galactosidase activity, whereas substitution of Lys300 with alanine had no effect on signal transduction. These phenotypes correlate with the inhibition of cAMP generation by direct cAMP measurement. Signal transduction was insensitive to pretreatment of cells with pertussis toxin, suggesting that a nonGi/Go pathway is activated by the EP3 receptor. Direct measurement of second messenger levels confirmed that there was no increase in cAMP levels mediated by the 77A splice variant, however, there was a modest increase in intracellular Ca2+. Partial blockade of the reporter activity with kinase inhibitors demonstrates that CRE activation is mediated in part by a Ca2+-dependent kinase pathway. These data suggest that the EP3 receptor signals through a novel cAMP response element binding protein/CRE pathway.

Prostaglandins (PGs) are ubiquitous lipid mediators derived from cyclooxygenase metabolism of arachidonic acid that exert a broad range of physiologic activities, including modulation of inflammation, ovulation and arterial blood pressure. PGE2, a chief cyclooxygenase product, modulates blood pressure and fertility, although the specific G protein-coupled receptors mediating these effects remain poorly defined. To evaluate the physiologic role of the PGE2 EP2 receptor subtype, we created mice with targeted disruption of this gene (EP2-/-). EP2-/- mice develop normally but produce small litters and have slightly elevated baseline systolic blood pressure. In EP2-/- mice, the characteristic hypotensive effect of intravenous PGE2 infusion was absent; PGE2 infusion instead produced hypertension. When fed a diet high in salt, the EP2-/- mice developed profound systolic hypertension, whereas wild-type mice showed no change in systolic blood pressure. Analysis of wild-type and EP2-/- mice on day 5 of pregnancy indicated that the reduced litter size of EP2-/- mice is due to a pre-implantation defect. This reduction of implanted embryos could be accounted for by impaired ovulation and dramatic reductions in fertilization observed on day 2 of pregnancy. These data demonstrate that the EP2 receptor mediates arterial dilatation, salt-sensitive hypertension, and also plays an essential part in female fertility.

G protein coupled receptors for prostaglandins are activated when agonists are bound to a binding pocket formed in part by the seven transmembrane domains. Recent studies have determined that substitution of a conserved threonine in the second extracellular loop of the prostaglandin EP3 receptor resulted in increased affinity for ligands with a C1 methyl ester moiety. The homologous threonine in the second extracellular loop of the human prostaglandin EP2 and EP4 receptors was mutated to alanine. When expressed in COS1 cells, detectable radioligand binding at both of these receptors bearing the threonine to alanine substitution (EP2T185A; EP4T168A) was abolished, as well as the receptors' ability to stimulate intracellular [cAMP]. In contrast, EP2 and EP4 receptors bearing conservative threonine to serine mutations (EP2T185S; EP4T168S) displayed Kd values for [3H]prostaglandin E2 similar to wild type receptors: 8.8 +/- 0.7 nM for EP2T185S compared to 12.9 +/- 1.2 nM for EP2 wild type; 2.0 +/- 0.8 nM for EP4T168S compared to 0.9 +/- 0.3 nM for the EP4 wild type receptor. The EC50 values for cAMP stimulation were 1.3 +/- 0.6 nM for EP2 wild type; 2.7 +/- 1.3 nM for EP2T185S; 1.1 +/- 0.3 nM for EP4 wild type; and 1.4 +/- 0.33 nM for EP4T168S. These studies suggest a critical role for the hydroxyl moiety on these conserved threonine residues at position 168/185 of the second extracellular loop in prostaglandin receptor-ligand interactions.

Prostaglandin E2 is the major cyclooxygenase product of arachidonic acid metabolism produced along the nephron. This autacoid interacts with four distinct, G-protein-coupled E-prostanoid receptors designated EP1-EP4. The intrarenal distribution of each receptor has been mapped and the consequences of receptor activation examined. EP3 receptor mRNA is expressed highly in the medullary thick ascending limb (mTAL) and collecting duct (CD). EP3 receptor activation inhibits cAMP generation via Gi, thus inhibiting vasopressin-stimulated water reabsorption in the CD. EP3 receptor activation also may contribute to PGE2-mediated inhibition of NaCl absorption in the mTAL. The EP1 receptor is coupled to increased cell [Ca2+]. EP1 mRNA expression is restricted to the CD, and receptor activation inhibits Na+ absorption. PGE2 also increases cAMP generation in the cortical thick ascending limb and CD; this may be due to EP4 receptor activation. EP4 mRNA is readily detected in the CD with little detectable EP2 expression. The EP4 receptor appears to be expressed both on luminal and basolateral membranes. EP4 receptor activation also may contribute to the regulation of renin release by the juxtaglomerular apparatus. The consequences of renal EP-receptor activation for salt and water balance may be determined by the relative renal expression of each of these receptors.

PGE2 exerts potent diuretic and natriuretic effects on the kidney. This action is mediated in part by direct inhibition of collecting duct Na+ absorption via a Ca++-coupled mechanism. These studies examine the role the Ca++-coupled PGE-E EP1 receptor plays in mediating these effects of PGE2 on Na+ transport. Rabbit EP1 receptor cDNA was amplified from rabbit kidney RNA. Nuclease protection assays demonstrated highest expression of EP1 mRNA in kidney, followed by stomach, adrenal, and ileum. In situ hybridization, demonstrated renal expression of EP1 mRNA was exclusively over the collecting duct. In fura-2-loaded microperfused rabbit cortical collecting duct, EP1 active PGE analogs were 10-1, 000-fold more potent in raising intracellular Ca++ than EP2, EP3, or EP4-selective compounds. Two different EP1 antagonists, AH6809 and SC19220, completely blocked the PGE2-stimulated intracellular calcium increase. AH6809 also completely blocked the inhibitory effect of PGE2 on Na+ absorption in microperfused rabbit cortical collecting ducts. These studies suggest that EP1 receptor activation mediates PGE2-dependent inhibition of Na+ absorption in the collecting duct, thereby contributing to its natriuretic effects.

The purpose of this study was to characterize the prostanoid receptors coupled to intracellular calcium in human erythroleukemia (HEL) cells, a cell line with platelet/megakaryocytic characteristics. Both prostaglandin E1 (PGE1) and iloprost increased cyclic AMP (cAMP) in HEL cells, but modulated [Ca2+]i by different mechanisms. Iloprost (10(-9) to 10(-6) M) had no effect on basal [Ca2+]i, but greatly potentiated the increase in [Ca2+]i produced by thrombin. This effect was mimicked by cholera toxin and other Gs-coupled receptors, and involved calcium influx since iloprost had no effect on [Ca2+]i in cells incubated in Ca2+-free buffer. Furthermore, iloprost did not increase the generation of baseline or thrombin-induced inositol phosphates at these concentrations. In contrast, PGE1 (10(-7) to 10(-5) M), but not iloprost, increased basal [Ca2+]i through a pertussis toxin-sensitive mechanism that involved stimulation of inositol phosphate generation and mobilization of intracellular calcium. The order of potencies of other prostaglandins that increased [Ca2+]i was not consistent with known IP, EP, DP, FP, or TP receptors. 11-Deoxy-16,16-dimethyl PGE2 was the most potent of the analogs tested (EC50 = 28 nM). In summary, at least two prostaglandin receptors are functionally coupled to intracellular calcium in HEL cells: a putative IP receptor coupled to Gs proteins that increases cAMP and enhances calcium influx, and a novel prostanoid receptor that evokes calcium mobilization through stimulation of phospholipase C by a pertussis toxin-sensitive pathway.

To elucidate roles of protein kinase C (PKC) zeta in rabbit kidney, PKC zeta was cloned from a rabbit kidney cortex cDNA library. Sequencing revealed a 2113 m insert with an open reading frame encoding a protein of 591 amino acids. The predicted amino acid sequence is 93.7% identical with rat PKC zeta. In situ hybridization in rabbit kidney with a riboprobe generated from the cloned cDNA, showed PKC zeta mRNA is highly expressed in proximal tubule, thick limb, and collecting duct. No message was detected over glomerular cells. Immunohistochemical studies using a monoclonal antibody against PKC zeta confirmed this distribution with low expression in vascular elements and high expression in tubule epithelium. Confocal microscopy showed diffuse cytosolic immunoreactivity in confluent cultured cortical collecting ducts (CCDs). However, in subconfluent cells, immunoreactivity was restricted to the peri-nuclear area. This differential distribution of PKC zeta in the CCD suggests that PKC zeta action be involved in growth and differentiation of the collecting duct. In conclusion, PKC zeta is differentially expressed in the rabbit kidney with high expression in the tubule epithelium and little expression in vascular elements. These studies suggest an important role for PKC zeta along the nephron.

The prostaglandin EP3 receptor binds Prostaglandin E2 in a ligand binding pocket formed in part by seven transmembrane alpha-helices. The present studies demonstrate that the second extracellular loop of the receptor is involved in prostanoid ligand recognition as well. Site-directed mutagenesis of seven conserved residues clustered in the amino portion of the second extracellular loop was performed. Receptors with single amino acid substitutions at each of these positions were transiently transfected into HEK293tsA201 cells, their ligand binding profiles assessed, and each receptor was tested for its ability to decrease intracellular cAMP levels. Substitution of Trp199 or Thr202 with alanine resulted in receptors with increases in affinity up to 128-fold for prostanoid compounds with a C1 methyl ester but wild type affinities for natural prostanoid ligands that have a carboxylate moiety at the C1 position. In contrast, substitution of Pro200 with serine caused a loss of selectivity up to 20-fold for naturally occurring prostanoid agonists as compared with the wild type EP3 receptor: the PS200 receptor displayed a decrease in affinity for E-ring compounds and an increase in affinity for F- and D-ring compounds. The EC50 for inhibition of cAMP remained unchanged for each receptor tested.

Expression of the rabbit EP3 receptor isoform 77A in COS1 and HEK293tsA201 cells demonstrated specific binding of [3H]prostaglandin (PG)E2 and receptor-evoked decreases in intracellular cAMP levels. Competition binding with PGE2, PGE2 methyl ester, misoprostol-free acid, misoprostol, and sulprostone suggested that a negative charge at the C1 position is essential for high affinity ligand binding and that the charge at this position is more important than steric bulk. Charged amino acid residues within the transmembrane (TM) domains of the receptor were mutated, and the resulting receptor proteins were analyzed for the effects of these mutations on receptor structure and/or function. Positively charged TM residues are candidates for interaction with the C1 carboxylic acid moiety of prostanoid ligands. Substitution of R329 (TM VII) with either alanine or glutamate resulted in a loss of both detectable [3H]PGE2 binding and receptor activation despite expression of the receptor protein as determined by immunoprecipitation and immunofluorescence. Substitution of K300 (TM V) with alanine had no effect on binding or signal transduction. Substitution of the conserved aspartic acid at position 338 (TM VII) with alanine caused a loss of detectable receptor-evoked inhibition of cAMP generation, although this mutation did not appreciably affect ligand binding. These studies suggest that R329 but not K300 is a key determinant in receptor/ligand interaction. Furthermore, D338 plays a critical role in G1 activation by the EP3 receptor.

Four prostaglandin E2 (PGE2) receptors designated EP1, EP2, EP3, and EP4 have been pharmacologically identified, cloned, and sequenced. The present studies determined the intrarenal distribution of these EP-receptor subtypes in human kidney using in situ hybridization with riboprobes for the human EP receptors. mRNA for the phosphatidylinositol hydrolysis-coupled EP receptor was highly expressed in cortical, outer medullary, and inner medullary collecting duct. RNA for the Gi-coupled EP3 receptor was primarily expressed in the cortical and outer medullary collecting duct, as well as in the medullary thick ascending limb; however, it was absent from the inner medullary collecting duct. Expression of mRNA for EP1 and EP3 in connecting segment could not be excluded. There was no expression of the GS-coupled EP2 receptor mRNA detected in human kidney by in situ hybridization; however, mRNA for the GS-coupled EP4 receptor was highly expressed in the glomerulus. These studies demonstrate distinct regions of intrarenal expression for the different EP receptors and suggest that each receptor subtype may modulate different aspects of renal function in humans.

Prostaglandin E2 (PGE2) is an important regulator of systemic hemodynamics and epithelial ion transport. To further investigate the mechanism of PGE2 action, a clone encoding a PGE2 receptor was isolated from a rabbit kidney cortex cDNA library. Expression of the full-length cDNA in COS-1 cells yielded a ligand-binding profile typical for a butaprost-insensitive Gs-coupled E-prostanoid (EP) receptor. Misoprostol-free acid, a receptor-selective PGE analogue, produced concentration-dependent increases in adenosine 3',5'-cyclic monophosphate production. The data are consistent with the receptor being an EP4 subtype. Ribonuclease protection assays demonstrated that this receptor gene is highly expressed in intestine, uterus, and thymus, with lower but significant expression in kidney, whole adrenal, lung, spleen, and stomach. In situ hybridization in kidney revealed intense hybridization to glomeruli and urothelium of the renal pelvis. This prostanoid receptor was also highly expressed in the duodenal epithelium and adrenal cortex. The tissue distribution suggests a functional role for this receptor in mediating glomerular effects of PGE2 and effects on aldosterone secretion, intestinal transport, and immune function.

The diverse intrarenal effects of the prostaglandins (PG) are mediated by distinct guanine nucleotide regulatory protein (G-protein)-coupled receptors. The cDNA for these receptors have been cloned, their signal transduction mechanisms determined, and their intrarenal distribution mapped. PGE2, the major intrarenal prostaglandin, interacts with at least three distinct E-prostanoid (EP) receptors that are highly expressed in specific regions of the kidney. Each EP receptor not only selectively binds PGE2, but also preferentially couples to different signal transduction pathways, including: stimulation of cAMP generation, via Gq (EP2 and EP4 receptors); inhibition of cAMP generation, via Gi (EP3 receptors); and activation of phosphatidylinositol hydrolysis (EP1 receptor), via one of the Gq family members. Activation of each these EP receptors is responsible for a distinct renal effect of PGE2, including its well-described renal hemodynamic and transport effects along the nephron. Other intrarenal prostanoid receptors include the PGF2 alpha receptor (FP), the thromboxane A2 receptor (TP) and the prostacyclin receptor (IP). Knowledge about localization of these receptors and their affinities for receptor-selective agonists and antagonists should aid in the understanding of renal disease and the development of therapeutic strategies for the use of these prostaglandin analogs in select renal diseases.

Endogenous prostaglandin (PG) E2 production potently modulates salt and water transport in the kidney. Multiple direct effects of PGE2 on epithelial water and sodium transport have been demonstrated in the rabbit cortical collecting duct (CCD). Both functional and molecular studies now suggest that these disparate effects of PGE2 on CCD function are mediated by different EP receptors. When added in the presence of vasopressin, PGE2 inhibits cyclic AMP generation and water absorption. These effects are mediated via an inhibitory G-protein (Gi). In situ hybridization demonstrates high levels of expression of the Gi-coupled EP3 receptor in the rabbit collecting duct. However, by itself, PGE2 also stimulates cyclic AMP generation and water permeability. These effects appear to be mediated via a distinct EP receptor (possibly an EP4 receptor). PGE2 also increases intracellular Ca2+ in the CCD and inhibits Na+ absorption via a Ca(2+)-dependent mechanism. The EP1 receptor is postulated to be responsible for this action of PGE2. We suggest receptor-selective prostaglandin analogs may be used to selectively modulate sodium and water transport in the kidney.

Four cDNA clones homologous with a murine prostaglandin E2 receptor have been isolated from a rabbit kidney cortex cDNA library. These cDNAs encode related proteins that differ only in their COOH-terminal sequences. Southern blot analysis of rabbit genomic DNA indicates that these receptor cDNAs represent alternatively spliced variants derived from a single gene. This was confirmed by isolation and sequence analysis of genomic clones containing common region exons and unique 3'-coding exons, which contained intron/exon boundaries at the predicted splice junctions. Transient expression of a novel full-length cDNA in COS1 cells confirmed the ligand binding profile typical of an EP3 receptor subtype. Ribonuclease protection assays indicate that the gene encoding these receptors is most highly expressed in kidney, adrenal, and stomach with lower but significant expression in uterus, lung, heart, ileum, spleen, and brain. Moreover, each of the cloned isoforms is expressed in the kidney. In situ hybridization analyses of rabbit kidney demonstrated that the level of expression is highest in the outer medulla with lesser expression in the cortex and no detectable expression in the inner medulla. The ligand binding profile and tissue distribution of these receptors is consistent with a functional role for this family of EP3 receptors in mediating the renal actions of prostaglandins as well as the effects of prostaglandins on gastric acid secretion and adrenal function.

The physiological effects of PGE2 appear to be mediated by at least three different "E-prostanoid" receptors designated EP1,EP2, and EP3. These receptors are differentially activated by structural PGE analogs (such as misoprostol) and each couples to a different signal transduction mechanism. Studies demonstrating that inhibition of water absorption in the collecting duct is mediated by a Gi coupled mechanism, suggests that an EP3 receptor is involved the renal effects of PGE2. We used in situ hybridization to determine the tissue distribution of the rabbit EP3 receptor. [alpha-35S] UTP labeled antisense RNA, comprising transmembrane domains IV through VII, was hybridized to tissue sections. Specific labeling of kidney, stomach and adrenal was observed. In the kidney, medullary thick ascending limb and cortical and medullary collecting ducts were intensely labeled, while no labeling of glomeruli, proximal tubules, or cortical thick ascending limbs was observed. The adrenal gland labeled exclusively in the medulla. In the stomach the gastric epithelial crypts were the predominant site of hybridization, without evidence of labeling of the smooth muscle. These results suggest an important role for the EP3 receptor in mediating PGE2 effects in these tissues.

Signaling through serotonin 5-HT1A receptors involves multiple pathways. We have investigated the functional coupling of the human 5-HT1A receptor to different G proteins using an in vitro reconstitution system based on the expression of recombinant receptor (r5-HT1A) and G alpha-subunits (rG alpha) in Escherichia coli. The r5-HT1A receptor was expressed by insertion in a vector allowing its active expression in E. coli inner membranes. Binding of the selective agonist [3H] +/- 8-hydroxy-(2-N-dipropylamine)tetralin ([3H]8-OH-DPAT) to intact bacteria or E. coli membranes was saturable with a KD of approximately 8 nM and an average of 120 sites/bacterium. Binding properties of several serotoninergic ligands to r5-HT1A receptors were comparable with those measured in mammalian cells. Incubation of rG alpha.beta gamma with E. coli membranes resulted in high affinity agonist [3H]8-OH-DPAT binding (KD = 0.7 nM) and titration with a panel of rG alpha subtypes showed the order of potency: rGi alpha-3 greater than rGi alpha-2 greater than rGi alpha-1 much greater than rGo alpha, while rGs alpha appeared incapable of interacting with 5-HT1A receptors. Moreover, agonist-mediated enhancement of [35S]guanosine 5'-O-(3-thiotriphosphate) binding to rGi alpha confirmed the achievement of the functional interaction between receptor and G proteins. Our findings are in agreement with the in vivo ability of 5-HT1A receptors to activate Gi alpha related to adenylyl cyclase inhibition or K+ channel activation, but do not support previously reported adenylyl cyclase stimulation through interaction with Gs alpha.

A novel in situ screening procedure was used to identify neutral mutations in the human beta 2 adrenergic receptor (beta 2AR). The coding region of the human beta 2AR gene was subcloned under transcriptional control of an inducible T7 promoter and used to transform Escherichia coli. Colonies expressing the beta 2AR bound the radiolabeled antagonist [125I]iodocyanopindolol and could be identified by autoradiography after transfer to nitrocellulose filters. The region of the beta 2AR between residues 76 and 83, in the second transmembrane helix, was mutagenized by a saturation mutagenesis technique, so that virtually all of the beta 2AR genes contained at least one mutation. Colonies retaining ligand binding activity were isolated using the in situ screen. Sequence analysis of the active mutant receptor genes allowed the identification of individual amino acid side chains which are essential for either ligand binding or structural integrity of the beta 2AR receptor.

Monoclonal antibodies reactive with distinct regions of the N-terminal domain of the lambda repressor protein have been isolated. By comparing the affinities of these antibodies for mutant repressors with increased and decreased thermal stabilities, each of the antibodies can be shown to bind to epitopes accessible in the native conformation of the N-terminal domain. Experiments probing antibody binding to protein fragments, mutant variants, and peptides have also been used to define likely regions of contact between the antibodies and the N-terminal domain.

Monoclonal antibody 51F recognizes determinants in the helix 4 region of the native form of the N-terminal domain of lambda repressor. A cassette mutagenesis method was used to introduce changes within this region, and antibody-reactive candidates were isolated and sequenced. The resulting data allow the identification of repressor side chains that are critical determinants of antibody binding. Four of these side chains are on the surface of the N-terminal domain and probably contact the antibody directly. These contact positions were then mutagenized individually, and the antibody binding phenotypes of a large number of singly mutant repressors were determined. Taken together, the mutational data allow a functional map of the recognition surface to be constructed and the physical nature of some of the specific interactions that stabilize the antibody-antigen complex to be surmised.

To learn about the V lambda gene segments that are expressed in lambda 3 light chains, the most recently identified lambda-chain subtype in inbred mice, we determined partial amino acid sequences of the V regions of two of these chains, L5-8 and Lc49 . The partial sequences were extended by establishing the complete V region sequence of cDNA for the lambda-chain mRNA from the hybridoma (RZ 5-8) and the myeloma ( CBPC -49) that produce these chains. The primer extension method used to sequence the cDNA is described in detail, because the same primer (a synthetic heptadecadeoxynucleotide ) can be used for sequencing cDNA for lambda-chains of all three subtypes of inbred mice and probably for lambda-chains from some other vertebrate species as well. The results confirm earlier preliminary findings that for both chains the V region is encoded by the V lambda 1 and J lambda 3 gene segments. The unmutated germ-line sequences of these gene segments are present in both chains, but the two chains, nevertheless, differ at codon 97, the V lambda-J lambda boundary. A T/G difference in the third position of this codon resulted in a codon for histidine (CAT) in one chain (L5-8) and for glutamine (CAG) in the other chain ( Lc49 ). This difference can be accounted for by variation in the site of V lambda 1-J lambda 3 recombination. Though the V region amino acid sequences of L5-8 and Lc49 differ only by the His/Gln substitution at position 97, the two chains have been shown (manuscript in preparation) to differ in their ability to form an effective combining site for the 2,4-dinitrophenyl group.