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Although many G-protein-coupled neurotransmitter receptors are potentially capable of modulating both voltage-dependent Ca(2+) channels (I(Ca)) and G-protein-gated K(+) channels (I(GIRK)), there is a substantial degree of selectivity in the coupling to one or other of these channels in neurons. Thus, in rat superior cervical ganglion (SCG) neurons, M(2) muscarinic acetylcholine receptors (mAChRs) selectively activate I(GIRK) whereas M(4) mAChRs selectively inhibit I(Ca). One source of selectivity might be that the two receptors couple preferentially to different G-proteins. Using antisense depletion methods, we found that M(2) mAChR-induced activation of I(GIRK) is mediated by G(i) whereas M(4) mAChR-induced inhibition of I(Ca) is mediated by G(oA). Experiments with the beta gamma-sequestering peptides alpha-transducin and beta ARK1(C-ter) indicate that, although both effects are mediated by G-protein beta gamma subunits, the endogenous subunits involved in I(GIRK) inhibition differ from those involved in I(Ca) inhibition. However, this pathway divergence does not result from any fundamental selectivity in receptor-G-protein-channel coupling because both I(GIRK) and I(Ca) modulation can be rescued by heterologously expressed G(i) or G(o) proteins after the endogenously coupled alpha-subunits have been inactivated with Pertussis toxin (PTX). We suggest instead that the divergence in the pathways activated by the endogenous mAChRs results from a differential topographical arrangement of receptor, G-protein and ion channel.

A cDNA encoding the rat mu-opioid receptor was expressed stably in a Rat-1 fibroblast cell line. Expression of this receptor was demonstrated with specific binding of the mu-opioid selective ligand [3H][D-Ala2,N-MePhe4,Gly5-ol]-enkephalin ([3H]DAMGO). In membranes of clone mu11 cells DAMGO produced a robust, concentration-dependent stimulation of basal high affinity GTPase activity. Cholera toxin-catalyzed [32P]ADP-ribosylation in membranes of this clone labelled a 40 kDa Gi family polypeptide(s) that was markedly enhanced by the addition of DAMGO. Antisera against Gi2alpha and Gi3alpha were both able to immunoprecipitate a [32P]-radiolabelled 40 kDa polypeptide(s) from DAMGO and cholera-toxin treated membranes of clone mu11, indicating that the mu-opioid receptor was able to interact effectively with both Gi2 and Gi3 in Rat-1 fibroblasts. A series of peptides derived from the delta-opioid receptor sequence were assessed for their ability to modify agonist-stimulated G protein activation and [3H] agonist binding to the receptor. In membranes from the clone mu11, specific binding of [3H]DAMGO was reduced by peptides corresponding to the NH2-terminal region of the third intracellular loop (i3.1) and the carboxyl-terminal tail (i4) of this receptor. Agonist stimulated GTPase activity and DAMGO dependent cholera toxin-catalyzed [32P]ADP-ribosylation were inhibited by peptides derived from the proximal (i3.1) and the distal portion (i3.3) of the third intracellular loop. Peptide i3.1 also inhibited DAMGO-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTP-gammaS) binding in the same membranes. In contrast, peptides derived from the second intracellular loop were without any effect.

The alpha2A-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of 'Gi-like' pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual Gi-family G-proteins the porcine alpha2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1alpha, Gi2alpha and Gi3alpha or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The alpha2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5'-[gamma-35thio]-triphosphate (GTP[35S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type Gi-like G-protein alpha-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of Gi1alpha, Gi2alpha and Gi3alpha resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed Gi-like G-proteins was resistant to pertussis toxin treatment. The Cys --> Gly mutations of Gi1alpha, Gi2alpha and Gi3alpha were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of Gi1alpha was correlated highly both with the level of expression of this G-protein and with the level of expression of the alpha2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase activity of the Cys --> Gly mutants of Gi1alpha, Gi2alpha and Gi3alpha required 10-15-fold higher concentrations of agonist than did stimulation of their wild-type counterparts, consistent with a model in which the affinity of functional interactions of the alpha2A-adrenoceptor with the wild-type G-protein is greater than with the pertussis toxin-resistant mutant G-protein.

The significance of Gi proteins for the physiological desensitization phenomena observed in brown-fat cells from cold-acclimated hamsters was investigated. For this purpose, pertussis toxin (the inhibitor of Gi function) was injected into control and cold-acclimated hamsters. After 3 days the thermogenic response to noradrenaline injection was monitored in the intact animals. It was found that the pertussis-toxin pretreatment did not affect the thermogenic response to noradrenaline. Nonetheless, the pertussis toxin pretreatment had a dramatic effect on the noradrenaline-sensitivity of isolated brown-fat cells (measured the following day as the respiratory response): a 250-fold-increased sensitivity to noradrenaline was observed in cells from control animals that had been pertussis-toxin pretreated. However, only a 20-fold increase was observed in cells from cold-acclimated hamsters, implying a lower complement of the Gi system in these cells. Therefore the content of Gi proteins was determined by quantitative immunoblotting of purified plasma-membrane proteins. Cold acclimation resulted in a nearly 50% reduction in the content of Gi 1 alpha and Gi 2 alpha, as well as of the beta-subunit, both when expressed on a protein basis and when related to the content of forskolin-stimulated adenylyl cyclase; when expressed per unit of [3H]ouabain-binding (NA+/K+-ATPase), the reduction was even higher. In view of the magnitude of the pertussis-toxin effect, it was concluded that Gi proteins must play a substantial role in the regulation of the response of brown-fat cells to noradrenaline. As the capacity of the Gi pathway is reduced rather than augmented during cold acclimation, Gi activity cannot be responsible for the desensitization to noradrenaline observed in cells from cold-acclimated animals. However, the reduced Gi content may explain the earlier observed desensitization to adenosine that occurs after acclimation to cold.

Prolonged exposure of alpha T3-1 pituitary gonadotrophs to a gonadotrophin-releasing hormone receptor agonist results in marked down-regulation of the pertussis toxin-insensitive G proteins Gq alpha and G11 alpha. The turnover of Gq alpha/G11 alpha was substantially accelerated in the presence of agonist. By contrast, the rate of degradation of the G protein Gi2 alpha was unaffected by agonist treatment. Analysis of Gq alpha/G11 alpha mRNA levels by reverse transcription-PCR demonstrated no detectable differences between control and agonist-treated cells. These studies indicate that gonadotrophin-releasing hormone receptor agonist-mediated down-regulation of Gq alpha/G11 alpha is a reflection of enhanced proteolysis of the activated G proteins.

The alpha 2-adrenergic receptors are linked to inhibition of adenylylcyclase and, under certain circumstances, to stimulation of phospholipid hydrolysis via pertussis toxin-sensitive G proteins. Here we show that alpha 2-adrenergic receptors can couple to an alternative signaling pathway. When expressed in Rat-1 cells, stimulation of the alpha 2A receptor, which couples to Gi2 and Gi3, causes rapid, transient activation of the protooncogene product p21ras as measured by an increase in the amount of bound GTP. Furthermore, alpha 2A receptor stimulation causes rapid phosphorylation of the p42 mitogen-activated protein (MAP) kinase. Pertussis toxin completely inhibits both p21ras activation and MAP kinase phosphorylation, but both responses appear to be independent of adenylylcyclase inhibition or phospholipase stimulation. Thus, alpha 2-adrenergic receptors can couple to the p21ras-MAP kinase pathway via Gi, which may explain the mitogenic potential of alpha 2 agonists in certain cell types; together with previous results, these findings further suggest that activation of this pivotal signaling pathway may be a common event in the action of Gi-coupled receptors.

Cholera toxin-catalysed [32P]ADP-ribosylations were performed in the absence of guanine nucleotides on membranes of a clone (1C) of Rat 1 fibroblasts which express high levels of the alpha 2-C10 adrenergic receptor. As well as incorporation of radioactivity into 45,000 and 42,000 M(r) polypeptides which represent forms of Gs alpha, there was weak labelling of a 40,000 M(r) polypeptide(s). Addition of the alpha 2 adrenergic agonist UK14304 to such assays enhanced markedly the incorporation of radioactivity into the 40,000 M(r) polypeptide(s) but did not alter labelling of the forms of Gs. We have previously demonstrated that the 40,000 M(r) polypeptide(s) labelled in such a manner represents a combination of the alpha subunits of Gi2 and Gi3 [Milligan et al. (1991) J. biol. Chem. 266, 6447-6455]. Mercuric acetate treatment of membranes prelabelled with [32P]ADP-ribose by exposure to pertussis toxin and [32P]NAD removed totally the radiolabel from both Gi2 and Gi3. However, cholera toxin-catalysed [32P]ADP-ribosylation of either the alpha subunits of the Gi-subtypes or of forms of Gs was unaffected by such treatment. By contrast, prolonged, but not short-term, exposure to neutral hydroxylamine removed radiolabel incorporated by cholera toxin from the Gi-subtypes and from Gs but did not remove [32P]ADP-ribose incorporated by pertussis toxin from the Gi-subtypes. It is concluded that ADP-ribosylation of pertussis toxin-sensitive G-proteins by cholera toxin, which can be induced by exposure of membranes to agonists for receptors which interact with that G-protein, is at an arginine residue. It is suggested that this residue may be Arg 179 in Gi2 alpha and Arg 178 in Gi3 alpha.

DNA encoding the human alpha 2-C-10-adrenergic receptor was transfected into Rat-1 fibroblasts by CaPO4 precipitation, and clones expressing the receptor were isolated and expanded. One clone (1C) expressing high levels of the receptor was studied in order to determine the contacts between this receptor and guanine nucleotide-binding proteins (G proteins) mediating second messenger signaling. The alpha 2-adrenergic agonist UK 14304 stimulated high affinity GTPase activity in membranes from these cells. Incubation of these membranes with Protein A-purified fractions from an antiserum able to identify the carboxyl-terminal decapeptide common to Gi1 alpha and Gi2 alpha was partially able to prevent agonist stimulation of high affinity GTPase activity. Similar results were produced with an antiserum that identifies the carboxyl-terminal decapeptide of Gi3 alpha. In contrast, equivalent fractions of antisera that identify the carboxyl-terminal decapeptides of Go alpha and Gs alpha did not inhibit receptor stimulation of high affinity GTPase activity. Coincubation of the membranes from the cells with Protein A-purified fractions from the anti-Gi1 alpha + Gi2 alpha antiserum and the anti-Gi3 alpha antiserum produced greater inhibition of UK14304-stimulated GTPase activity than did either of the two antisera in isolation. These data show direct interaction of the human alpha 2-C10-adrenergic receptor, when expressed in this clone of Rat-1 fibroblasts, with multiple pertussis toxin-sensitive G proteins and demonstrate that a single receptor has the physical capacity to interact functionally with more than a single pertussis toxin-sensitive G protein in a native membrane. Furthermore, because the two antisera were able to inhibit receptor stimulation of high affinity GTPase activity to similar degrees, the G protein pools identified by these antisera must contribute similar amounts of the total receptor activation of pertussis toxin-sensitive G proteins in these cells.

A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of [3H]yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study. When cholera toxin-catalyzed ADP-ribosylation was performed with [32P]NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha. Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells. Dose response curves for U.K.14304 enhancement of cholera toxin-labeling of the 40-kDa polypeptide(s) and stimulation of high affinity GTPase activity were identical. By contrast, U.K.14304 was ineffective in either assay in membranes from the 4D cells, demonstrating this effect to be dependent upon receptor activation. Furthermore, the alpha 2 receptor antagonist yohimbine blocked all effects of U.K.14304. The agonist promotion of cholera toxin-catalyzed ADP-ribosylation of Gi was completely blocked by guanine nucleotides. Whether GDP or GDP + fluoroaluminate (as a mimic of GTP) was used, blockade of the agonist effect was complete and indeed both conditions prevented agonist-independent labeling by cholera toxin of the 40-kDa polypeptide(s). Mg2+ produced an agonist-independent cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s) but even in the presence of [Mg2+], agonist-stimulation of cholera toxin-labeling of the 40-kDa polypeptide(s) was observed and was additive with the effect of [Mg2+]. Agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi was completely attenuated by pretreatment of the cells with pertussis toxin, which prevents contact between receptors and G-proteins which are substrates for this toxin. By contrast, pretreatment of the cells with concentrations of cholera toxin able to "down-regulate" essentially all of the membrane-associated Gs alpha did not prevent agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular proliferation of rat glioma C6 BU1 cells in tissue culture is dependent on the presence of either calf or foetal-calf serum in the medium. Foetal-calf serum stimulated a high-affinity GTPase in membranes derived from C6 BU1 cells. Pretreatment of the cells with pertussis toxin decreased the high-affinity GTPase activity substantially, and attenuated the foetal-calf-serum-stimulated increase in this GTPase activity. Cholera toxin, in contrast, did not modulate the response to foetal-calf serum. Foetal-calf serum did not inhibit adenylate cyclase activity in membranes of these cells, indicating that the G-protein that was stimulated by foetal-calf serum was not Gi (the inhibitory one). Although the nature of the specific component of foetal-calf serum responsible for this pertussis-toxin-sensitive receptor-mediated stimulation of high-affinity GTPase activity has not been identified, it was mimicked neither by bombesin, which can stimulate inositol phospholipid turnover via a guanine nucleotide binding protein, nor by platelet-derived growth factor, which is present in substantial concentrations in foetal-calf serum. This report represents the first demonstration of a pertussis-toxin-substrate-mediated response in this cell line and provides further evidence that G-proteins other than Gi can be functionally inactivated by pertussis toxin.

The pertussis toxin substrate from RAW264 macrophage cell membranes was characterized by two-dimensional gel electrophoresis and by immunoblots using antibodies directed against different guanine nucleotide binding proteins. RAW264 membranes were found to contain one major pertussis toxin substrate, which was recognized by both antibodies AS/6 and LE/3. The AS/6 antibody was made against a synthetic peptide corresponding to the carboxyl-terminal decapeptide of the alpha-subunit of transducin, and the LE/3 antibody was made against the peptide corresponding to amino acids 160-169 of a guanine nucleotide binding protein (Gi-2-alpha) cloned from a mouse macrophage cell line. The RAW264 pertussis toxin substrate was not recognized by either antibody CW/6 or antibody RV/3, which recognize the 41-kilodalton alpha-subunit of brain Gi (Gi-1-alpha) and Go-alpha, respectively. Pertussis toxin substrates from bovine brain were resolved into four major alpha-subunits by two-dimensional gel electrophoresis, and the LE/3 antibody recognized only one of the four proteins. The brain LE/3 reactive protein also reacted with the AS/6 antibody, migrated with a 40K molecular weight, and had an isoelectric point slightly more basis than the RAW264 pertussis toxin substrate. Therefore, the major pertussis toxin substrate in RAW264 cells appears to be Gi-2, and bovine brain contains a relatively minor amount of a closely related guanine nucleotide binding protein.

Amounts of the guanine nucleotide binding regulatory proteins which are also pertussis toxin substrates (such as Ni and No) were measured in rat glioma, C6BU-1, cells and in neuroblastoma X glioma, NG108-15, hybrid cells. Measurements were performed both by quantitating pertussis toxin catalyzed ADP-ribosylation and by quantitative immunoblotting with affinity purified antibodies specific for Ni or No. The amounts of pertussis toxin substrate in C6 and NG108-15 cells are 7.5 and 0.6 pmol/mg membrane protein, respectively. These levels are minimum values and higher estimates of the total amounts of N proteins in the two cells are obtained by quantitative immunoblot analysis of the beta-subunit common to all N proteins. Immunoblots with specific antibodies show that NG108-15 cells contain 3.8 pmol/mg of No and detectable but small (less than 0.1 pmol/mg) amounts of Ni. In contrast, C6 cell membranes contain no detectable No and only 0.14 pmol/mg Ni. Thus, C6 cells contain large amounts of a pertussis toxin substrate which is neither Ni nor No.

Cholera and pertussis toxin-sensitive G-proteins were examined using specific immunological probes in wild type NIH3T3 cells and in clones of these cells containing the N-ras gene attached to a promotor where expression either was (T15+) or was not (T15-) induced. The major pertussis toxin sensitive-polypeptide had the immunological characteristics of Gi2. Two distinct forms of Gs alpha (45 and 42 kDa) were identified. Long term over-expression of p21N-ras (T15+ cells) did not alter the levels of Gi2 alpha or of Gs alpha. Pretreatment of NIH3T3 or T15 cells with either pertussis toxin or cholera toxin led to the complete in situ ADP-ribosylation of the respective G-proteins. Modification of Gi2 by pertussis toxin, however, had no inhibitory effect on the ability of bombesin to stimulate the production of inositol phosphates in any of these cells lines. Treatment of these cells with cholera toxin elicited a potent inhibition of the bombesin-stimulated production of inositol phosphates. This could be mimicked, however, by other agents which increase intracellular cyclic AMP concentrations. Cholera toxin treatment did not produce a significant alteration in the number of bombesin receptors on the cell surface. These results suggest that, in the T15 cell line, enhanced coupling of bombesin receptors to a phospholipase C-mediated hydrolysis of inositol phospholipids is either produced directly by p21N-ras or that overexpression of this gene product leads to the enhanced expression or function of a cholera and pertussis toxin-insensitive G-protein which then mediates the effect.

Rat glioma C6 BU1 cells contain a pertussis toxin substrate of 40 kDa which does not appear to be identical with Gi,Go or transducin. The GTP analogue, GTP[gamma S], inhibited the rate of pertussis toxin-catalysed ADPribosylation of this protein, while the GDP analogue GDP[beta S] stimulated this reaction. A protein of the same kDa value was ADPribosylated by cholera toxin in the absence of added guanine nucleotides. It is suggested that this 40 kDa protein can be a substrate for both cholera and pertussis toxins under appropriate conditions.

Antisera raised against the carboxy-terminal decapeptide (KENLKDCGLF) of transducin-alpha detected the 40 kDa, major pertussis toxin substrate of human neutrophils. The antisera also detected this protein in undifferentiated HL-60 and U937 cells, and revealed an approx. 2-fold increase in protein/mg membrane protein with differentiation into mature phagocytic cells. The results provide direct immunochemical evidence for the presence of a novel, pertussis toxin-sensitive guanine nucleotide-binding protein in human leukocytes.

Pertussis toxin catalyzes incorporation of 20.2 pmol of ADP-ribose/mg of protein into approximately 40-kDa protein(s) in human neutrophil membranes compared with 14.1 pmol/mg in bovine brain membranes. Based on these measurements we estimate that pertussis toxin substrate(s) should represent at least 0.085% of total membrane protein in neutrophils. Both brain and neutrophil membranes show high concentrations (0.34 versus 0.16% of total membrane protein, respectively) of the common beta subunit of guanine nucleotide binding proteins. Affinity purified antibodies specific for Go-alpha fail to detect any protein in immunoblots of neutrophil membranes (150 micrograms) under conditions where as little as 10 ng of purified Go-alpha is detectable, and Go-alpha is readily detected in brain membranes (100 micrograms). An antiserum against transducin that cross-reacts strongly with Gi-alpha, detects as little as 5 ng of purified Gi-alpha and readily detects Gi-alpha in brain membranes, but in neutrophil membranes, the antiserum detects an approximately 40-kDa band that corresponds to less than 10% of the expected amount of pertussis toxin substrate(s). The results show that human neutrophil membranes contain relatively large amounts of pertussis toxin substrate(s), but that the predominant pertussis toxin substrate is immunochemically distinct from previously identified substrates, transducin, Gi, and Go.

The anti-helminthic drug suramin inhibited the basal high-affinity GTPase activity of both C6 BU1 glioma and NG 108-15 neuroblastoma x glioma hybrid-cell membranes with an IC50 (concentration causing half-maximal inhibition) value close to 30 micrograms/ml. This effect was shown to occur via a non-competitive mechanism in which the binding affinity of the G-proteins for GTP was not altered, but the maximal velocity of the subsequent hydrolysis was reduced. In NG 108-15 membranes, both opioid peptides and foetal-calf serum stimulated high-affinity GTPase activity in a pertussis-toxin-sensitive manner. These effects have previously been shown to be mediated by different G-proteins [McKenzie, Kelly, Unson, Spiegel & Milligan (1988) Biochem. J. 249, 653-659]. Suramin completely prevented the opioid-peptide-stimulated increase in GTP hydrolysis, but did not prevent the opioid peptide from binding to its receptor. Suramin, however, did not block the foetal-calf-serum-stimulated GTPase response. This selective action of suramin provides further evidence for distinct roles for two separate pertussis-toxin-sensitive G-proteins in signal transduction in NG 108-15 membranes and provides the first evidence for a selective effect of a drug on the functions of different G-proteins.

We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG108-15 cells. These antibodies attenuated opioid-stimulated high-affinity GTPase activity, but did not markedly affect the response to foetal-calf serum. We conclude that receptors for the opioid peptides function via the inhibitory G-protein (Gi), whereas foetal-calf serum activates a second pertussis-toxin-sensitive G-protein, which has a C-terminal sequence significantly different from that of Gi.

1 Fusion proteins were constructed between the human 5-HT(1A) receptor and pertussis toxin-resistant forms of both G(i1)alpha and G(o1)alpha mutated at residue(351) from cysteine to either glycine or isoleucine. Each of these was expressed stably in HEK293 cells. 2 Increasing concentrations of GDP inhibited binding of the agonist [(3)H]-8-OH-DPAT but not the antagonist [(3)H]-MPPF to each construct. 3 The IC(50) for GDP was greater for constructs containing isoleucine at residue(351) of the G proteins compared to those with glycine at this position. 4 The G protein antagonist suramin had similar effects to GDP on the binding of [(3)H]-8-OH-DPAT. 5 The proportion of 5-HT(1A) receptor binding sites detected by [(3)H]-MPPF that displayed high affinity for 8-OH-DPAT was significantly greater when the interacting G protein contained isoleucine rather than glycine at residue(351). 6 The 5-HT(1A) receptor displayed similar avidity of interaction with G(i1)alpha and G(o1)alpha. 7 These results indicate that a higher avidity ternary complex is formed between 8-OH-DPAT, the 5-HT(1A) receptor and G proteins when isoleucine rather than glycine is located at residue(351) of the interacting G protein. [ABSTRACT FROM AUTHOR]

To assess the relative capacity of the human δ opioid receptor to activate closely related G proteins, fusion proteins were constructed in which the α-subunits of either G[sub i1] or G[sub o1], containing point mutations to render them insensitive to the actions of pertussis toxin, were linked in-frame with the C-terminus of the receptor. Following transient and stable expression in HEK 293 cells, both constructs bound the antagonist [[sup 3]H]naltrindole with high affinity. d-ala[sup 2],d-leu[sup 5] Enkephalin effectively inhibited forskolin-stimulated adenylyl cyclase activity in intact cells in a concentration-dependent, but pertussis toxin-insensitive, manner. The high-affinity GTPase activity of both constructs was also stimulated by d-ala[sup 2],d-leu[sup 5] enkephalin with similar potency. However, enzyme kinetic analysis of agonist stimulation of GTPase activity demonstrated that the GTP turnover number produced in response to d-ala[sup 2],d-leu[sup 5] enkephalin was more than three times greater for G[sub i1]α than for G[sub o1]α. As the effect of agonist in both cases was to increase V[sub max] without increasing the observed K[sub m] for GTP, this is consistent with receptor promoting greater guanine nucleotide exchange, and thus activation, of G[sub i1]α compared with G[sub o1]α. An equivalent fusion protein between the human µ opioid receptor-1 and G[sub i1]α produced a similar d-ala[sup 2],d-leu[sup 5] enkephalin-induced GTP turnover number as the δ opioid receptor-G[sub i1]α fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of G[sub i1]α. [ABSTRACT FROM AUTHOR]