The active site of human C4a anaphylatoxin.

The human C4 activation peptide C4a has recently been shown to be biologically active and to share common tissue receptors with human C3a anaphylatoxin. Human C3a and C4a each induce contraction and cause cross-desensitization of isolated guinea-pig ileal strips. The essential active site of C3a is comprised in the model peptide containing the five COOH-terminal residues, Leu-Gly-Leu-Ala-Arg. The anaphylatoxic activities of the corresponding C4a pentapeptide, Ala-Gly-Leu-Gln-Arg, and several other synthetic peptides related to the COOH-terminal sequence of human C4a were examined. The C4a pentapeptide induced contraction of guinea-pig ileum at 1 X 10(-3) M and produced a wheal and flare reaction in human or guinea-pig skin when 2-5 mumols were injected intradermally. The corresponding C3a pentapeptide is 500-fold more active, since it induces contraction of guinea-pig ileum at 3-4 X 10(-6) M and only 4-10 nmole induce a visible skin reaction. Although the C4a pentapeptide is relatively inactive compared to the C3a pentapeptide, two analogs of these peptides, Leu-Gly-Leu-Gln-Arg and Ala-Gly-Leu-Ala-Arg, each exhibited significantly greater activity than Ala-Gly-Leu-Gln-Arg and each analog desensitized ileal smooth muscle towards contraction by either C3a or C4a. Thus it is a combination of two amino acid substitutions, the Ala for Leu-73 and Gln for Ala-76, in the COOH-terminal pentapeptide of C3a that accounts for the markedly reduced activity of C4a. The contribution of the COOH-terminal portion of C4a on its activity was further documented by examining the C4a octapeptide, Lys-Gly-Gln-Ala-Gly-Leu-Gln-Arg and a trialanyl analog, Ala-Ala-Ala-Ala-Gly-Leu-Gln-Arg. The C4a octapeptide, C4a (70-77), exhibited 5-fold greater biologic activity than the C4a pentapeptide, while the trialanyl analog was 40-fold more active. Anaphylatoxic activities of the C4a-(73-77) pentapeptide, C4a-(70-77) octapeptide, and the trialanyl octapeptide analog and their ability to specifically block the action of C3a and C4a on smooth muscle tissue support the conclusion that, as in C3a, the essential active site of C4a resides at its COOH terminus. Since C4a functions as an anaphylatoxin and significant quantities of this mediator may be generated in individuals with hereditary angioneurotic edema (HANE), the hypotheses that the kinin-like activity promoting edema in HANE patients is derived solely from component C2 and/or kininogens should be reappraised. The activities previously assigned to C4a and now confirmed by synthetic C4a analog peptides suggest that the kinin-like activity generated in HANE plasma may be derived in part from C4a.

Antigenic specificity of a rabbit antiserum raised against the 15-28 segment of thymosin alpha 1.

Thymosin alpha 1, an acidic 28-residue peptide, enhances immune function. We have described a radioimmunoassay for this thymic factor based on a rabbit antiserum raised against a thymosin alpha 1-(15-28) conjugate (Incefy et al., J. Immun. Meth. 1986, in press). The detailed antigenic specificity of this antiserum was determined by measuring the ability of synthetic segments and analogues of thymosin alpha 1 and related peptides to compete with radioiodinated Ac-Tyr-thymosin alpha 1-(15-28) in this radioimmunoassay. The antiserum bound segments Ac-(1-28), (15-28), (20-28) and (21-28) with nearly equal efficiency but failed to bind segments Ac-(1-10), (11-20), (19-24) and (22-28). Thus, the major immunoreactive site seen by the antiserum is the COOH-terminal segment (21-28) (Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH). Immunoreactivity of (21-28) was nearly abolished when the carboxylate groups of Glu-21, Glu-27 and Asn-28 were omitted separately. The antiserum bound to prothymosin alpha and thymosin alpha 11, which lack the alpha-carboxylate group of Asn-28, with 0.9 and 0.2%, respectively, of the efficiency of thymosin alpha 1. But it bound nonspecifically to parathymosin alpha, which contains the internal segment . . . -Glu-Val-Val-Glu-Glu-Glu-Glu-Asn- . . . . Residues Glu-21, Glu-27 and Asn-28 of thymosin alpha 1 may be important features of the antigenic site through their ability to induce helical structure, through the ability of their negatively charged carboxylate groups to bind to specific sites on the antibody or both.

Distribution of glucagon receptors on hormone-specific endocrine cells of rat pancreatic islets.

Glucagon is insulinotropic, but it remains uncertain whether the insulinotropic action is mediated directly by glucagon receptors expressed on beta-cells or by cross-binding to the insulinotropic glucagon-like peptide-1 (GLP-1) receptor known to be expressed on beta-cells. Binding of [125I]glucagon to GLP-1 receptors and not to glucagon receptors has been reported in tumor-derived beta-cells (15). The objectives of the current study were to use receptor-binding techniques and a glucagon receptor-specific antiserum to determine whether glucagon receptors are present on beta-cells. Specific binding (7.2 +/- 0.8%) of [125I]GLP-1 to beta TC-3 cells was displaced equivalently with GLP-1 and exendin-(9-39) )Kd = 0.9 and 0.4 nM. respectively), whereas approximately 700-fold higher concentrations of glucagon were required for equal displacement (Kd = 400 nM). Binding of [125I]glucagon to beta TC-3 cells (approximately 1%) was displaced equivalently with 1 microM glucagon, GLP-1, or exendin-(9-39). These observations support earlier findings that beta TC-3 cells do not express functional glucagon receptors. However, specific binding of [125I]glucagon was observed on INS-1 cells (2.3 +/- 0.2%); this was displaced with glucagon (Kd = 1 nM), but not 1 microM GLP-1 or exendin-(9-39). To examine the distribution of glucagon receptors on native beta-cells, dispersed cultured rat islets were immunostained for glucagon, somatostatin, or insulin in combination with a polyclonal rabbit antiserum raised to an extracellular portion of the glucagon receptor (KD-14). The glucagon receptor antiserum colocalized staining with approximately 97% of immunoreactive insulin cells, 9% of immunoreactive glucagon cells, and 11% of immunoreactive somatostatin cells. Perfusion of the rat pancreas with concentrations of glucagon as low as 10(-12) M resulted in significant insulin release. These results suggest that whereas the tumor-derived beta-cell line beta TC-3 does not express functional glucagon receptors, INS-1 cells and isolated rat pancreatic beta-cells have specific glucagon receptors, as do a subpopulation of alpha- and delta-cells. A model is proposed for the role of glucagon in islet hormone secretion during feeding and fasting.

Selective deficiency of guanine nucleotide-binding protein Go in two dopamine-resistant pituitary tumors.

G proteins were quantitated by immunoblot in normal rat anterior pituitary, an estrone-induced pituitary adenoma, and in two transplantable pituitary tumors resistant to dopamine, 7315a and MtTW15. Antisera specific for the alpha o and beta subunits or the alpha i subunit of G proteins were tested with all preparations. While the alpha i and beta subunits were found to be present in variable concentrations in all preparations, the alpha o subunit was very low or undetectable in the transplantable tumors. These findings suggest that tumor resistance to the dopamine inhibitory actions on prolactin release and on tumor growth may be due to the deficiency of a Go protein.

Receptor and effector interactions of Gs. Functional studies with antibodies to the alpha s carboxyl-terminal decapeptide.

Antibodies generated to a synthetic decapeptide, RMHLRQYELL, representing the carboxyl-terminus of Gs-alpha have been characterized in immunoblots and functional studies. This antibody, designated RM, reacts exclusively with a doublet of proteins of 52 and 45 kDa in immunoblots of bovine brain and wild-type S49 murine lymphoma cell membranes. No such reactivity is seen in membranes from cyc- S49 cells, which lack Gs. RM blocks receptor-mediated activation of Gs and adenylyl cyclase in membranes from wild-type S49 cells. RM could also immunoprecipitate adenylyl cyclase activity in detergent extracts from GTP[gamma]S- or fluoride-preactivated bovine brain membranes; thus binding of alpha s to effector and carboxyl-terminal antibody was mutually compatible. Such experiments provide an approach for the elucidation of functionally relevant interactions of G-proteins with receptors and effectors in the membrane.

Antibodies as probes of G-protein receptor-effector coupling and of G-protein membrane attachment.

The specificity of antisera raised against synthetic decapeptides corresponding to the C-terminus of G-protein alpha-subunits was rigorously defined. Antisera raised against alpha-subunit C-terminal decapeptides proved capable of immuno-precipitating their cognate G-proteins, as well as recognizing these proteins in native cell membranes. Thus the alpha s-specific antiserum could block agonist-stimulated adenylyl cyclase activity in native membranes and also immuno-precipitate an activated alpha s-adenylyl cyclase complex. The alpha i 2-, but not alpha i 3- and alpha z-specific antiserum could block agonist-mediated inhibition of adenylyl cyclase in human platelet membranes. These results indicate that the C-terminal decapeptide is involved in G-protein receptor, but not effector, coupling. These antisera also proved useful in immunoprecipitation of endogenous and transfected alpha-subunits in COS cells. Using this approach, we were able to show that both alpha s and alpha i are membrane associated, but only the latter is myristylated. A mutant alpha i 1 (second residue Gly changed to Ala) fails to incorporate myristate and is localized in the soluble fraction. Myristylation is thus essential for membrane attachment of alpha i, but not alpha s.

A radioimmunoassay for thymosin alpha-1.

A new radioimmunoassay (RIA) is described for the quantitation of thymosin alpha-1 (alpha-1). The assay employs an antiserum specific for the COOH-terminal half segment 15-28 of alpha-1, synthetic alpha-1-(15-28) as the hormone standard, and a radioiodinated N alpha-acetyltryrosyl-alpha-1-(15-28) as the tracer. Since alpha-1-(1-28) lacks a phenolic ring for direct radioiodination, the N alpha-acetyltyrosyl-alpha-1-(15-28) was synthesized by the solid-phase method. The peptide bears a Tyr in place of Lys in position 14 of the natural peptide. It showed full alpha-1-(15-28) immunoreactivity and its radioiodinated derivative served as tracer in the RIA. An anti-alpha-1-(15-28) antiserum was raised in a rabbit and was shown to recognize alpha-1-(15-28) or its tyrosyl analogue, and the peptide, alpha-1-(1-28). But it did not recognize other thymic hormones or the biologically active segment 32-36 of thymopoietin, or structurally unrelated peptides. It could also detect natural alpha-1 cross-reacting material in the cytoplasm of cultured human thymic epithelial cells as measured by indirect immunofluorescence. In the RIA, as little as 9 pg of alpha-1-(15-28) equivalents in a 50 microliter sample could be detected. In addition, alpha-1-(1-28)-like immunoreactivity was quantitated in 6 human thymus homogenates and ranged from 0.5 to 4.5 ng/mg of protein.

Biological activities of des-His1[Glu9]glucagon amide, a glucagon antagonist.

Hyperglycemia in diabetes mellitus is generally associated with elevated levels of glucagon in the blood. A glucagon analog, des-His1[Glu9]glucagon amide, has been designed and synthesized and found to be an antagonist of glucagon in several systems. It has been a useful tool for investigating the mechanisms of glucagon action and for providing evidence that glucagon is a contributing factor in the pathogenesis of diabetes. The in vitro and in vivo activities of the antagonist are reported here. The analog bound 40% as well as glucagon to liver membranes, but did not stimulate the release of cyclic AMP even at 10(6) higher concentration. However, it did activate a second pathway, with the release of inositol phosphates. In addition, the analog enhanced the glucose-stimulated release of insulin from pancreatic islet cells. Of particular importance were the findings that the antagonist also showed only very low activity (less than 0.2%) in the in vivo glycogenolysis assay, and that at a ratio of 100:1 the analog almost completely blocked the hyperglycemic effects of added glucagon in normal rabbits. In addition, it reduced the hyperglycemia produced by endogenous glucagon in streptozotocin diabetic rats. Thus, we have an analog that possesses properties that are necessary for a glucagon antagonist to be potentially useful in the study and treatment of diabetes.

Differential expression of low molecular weight form of Gs-alpha in neostriatum and cerebellum: correlation with expression of calmodulin-independent adenylyl cyclase.

Adenylyl cyclase of neostriatal plasma membranes is far less responsive to stimulation by Ca2+/calmodulin than is the enzyme from cerebellum. When these tissues were investigated with antibodies against known G-proteins, no difference could be detected in any G-protein species, other than Gs. Whereas the cerebellum expressed predominantly the large form of Gs-alpha, the neostriatum expressed mainly lower molecular weight forms. The possibility is considered that there is selective association between lower molecular weight forms of Gs and calmodulin-independent forms of adenylyl cyclase.

Nonadherence and osteoporosis treatment preferences of older women: a qualitative study.

BACKGROUND: Physicians must have an understanding of patients' medication beliefs in order to enhance medication adherence. To increase understanding, this study examined how beliefs about medication and four osteoporosis treatments influenced treatment selection and adherence. METHODS: Six focus groups, three with 28 African Americans and one with 11 non-Hispanic white women, were conducted in English. Two groups with 16 Hispanics were conducted in Spanish. The convenience sample was recruited from senior centers and housing in lower socioeconomic geographic areas. The average age was 74.8 +/- 1.1 years. RESULTS: Adherence was associated with recognition of the serious consequences of nonadherence, realization of the beneficial effects, and the belief that medicines are not harmful. Doubts about physicians' competence to prescribe appropriate drugs were also revealed. Women who thought they were unlikely to fracture or perceived fracture outcomes as not severe chose no treatment. If they identified a need, they weighed benefits against the attendant risks to find the best alternative among the affordable options. Price considerations eliminated raloxifene and alendronate. Consideration of side effects eliminated estrogen and raloxifene. Calcium was viewed as a low-cost, low-risk alternative. Those who could afford alendronate and who viewed its side effects as preventable preferred it. Benefit and risk assessments may have been biased by fear of cancer and thromboembolic events. CONCLUSIONS: Women's beliefs about necessity of treatment, medication safety, cost of treatment, and treatment goals appear critical to osteoporosis treatment selection and adherence.

The effects of knowledge, attitudes, and significant others on decisions to enroll in a clinical trial on osteoporosis: implications for recruitment of older African-American women.

This preliminary study explored the roles of knowledge, attitudes, and significant others on decisions of older African-American women to enroll in a clinical trial involving estrogen and osteoporosis. Sixteen older African-American women (average age 75 years) participated in three focus groups. Twelve of the women had enrolled in the clinical trial and four, although eligible, refused to enroll. Discussions revealed that knowledge of osteoporosis and estrogen and expectations of personal rewards and group benefits from medical research appear to differentiate the women who participated in the clinical trial from those who refused. The women who participated also perceived the research institution as accessible. In addition, assuring full disclosure of testing procedures and test results eased their apprehensions about participation. However, the women who refused to enroll saw no personal benefit and were unwilling to expose themselves, in part because of their age, to the risks of taking estrogen and the uncertain outcomes of the clinical trial. The study illustrates how focus groups can be used to develop multiple strategies to enable recruitment of older African-American women with different demographic characteristics, levels of knowledge, and attitudes toward a disease and medical research.

Synthesis of 2-methyl and 2-carboxymethyl derivatives of ornithine and arginine.

Synthesis and characterization of a dozen derivatives of ornithine and arginine bearing 2-methyl or 2-carboxymethyl substituents are described. These substituents were introduced by dilithiation of 3-(4-methoxybenzylidineamino)-2-piperidinone with lithium diisopropylamide followed by regiospecific alkylation at C-3 with iodomethane or ethyl bromoacetate. 2-Methyl-D,L-ornithine was obtained in three steps from 3-amino-2-piperidinone in 68% overall yield, and 2-carboxymethyl-D,L-ornithine was isolated in 56% overall yield. 2-Methyl- and 2-carboxymethyl-D,L-arginine were obtained by mild acid hydrolysis to remove the 4-methoxybenzylidine group, N-acylation with 4-toluenesulfonyl chloride, mild alkaline hydrolysis of the lactam ring and the ester group, guanidination of the 5-amino group with O-methylisourea, and strong acid hydrolysis to remove the 4-tolunenesulfonyl group. Several of these compounds are inhibitors of carboxypeptidase B.

Identification of an essential serine residue in glucagon: implication for an active site triad.

Several glucagon analogs containing substitutions for serine have been synthesized to assess the role of the four serine residues in the hormone. The strategic importance of His1 has been confirmed, and we have previously identified an aspartic acid critical for activity at position 9. While these findings have led to a series of pure glucagon antagonists, the details of specific glucagon-receptor interactions that switch on the ensuing signaling events are still not readily apparent. The requirement for serine was tested by the chemical synthesis of a series of analogs containing substitutions for the hydrophilic hydroxyl group in each of the highly conserved serine residues at positions 2, 8, 11, and 16 of glucagon. The resulting analogs were analyzed in rat hepatocyte membranes for their receptor-binding affinities as well as their abilities to stimulate adenylate cyclase. Positions 2 and 8 were the most sensitive to modification, where both binding and activity were adversely affected. This is consistent with the notion that although the sequence responsible for transduction lies in the amino-terminal half of glucagon, some residues at that end also contribute to binding affinity. Modifications at position 11 generated high-binding-affinity derivatives that were full or moderate agonists. In contrast, position 16 replacement analogs maintained significant receptor binding affinities while the agonist properties were almost completely lost, thus separating binding and transduction functions. Therefore, Ser16 is a third critical residue that determines glucagon activity. It is postulated, but not proven, that a serine residue, together with His1 and Asp9, may participate in the putative active center of glucagon, which, upon initial recognition and binding to receptor, leads to transduction of the biological signal. A dependence of the glucagon action on a three-residue cooperative mechanism might be analogous to the charge-relay scheme of serine proteases. It is suggested that, after binding to its receptor, glucagon becomes activated and functions like a coenzyme in catalyzing the specific hydrolysis of a peptide bond in the receptor, generating new amino and carboxyl end groups, and that one of these exposed chains may contact the GTP-binding protein and activate it for further interaction with adenylate cyclase. This idea was supported by inhibition experiments with 4-amidinophenylmethanesulfonyl fluoride (APMSF), a specific and irreversible inhibitor of serine proteases, which at a concentration of 5 mM completely suppressed cAMP formation by glucagon in liver membranes. cAMP formation was not affected if either glucagon or membranes were separately pretreated with APMSF and then assayed.

Persistent activation of the alpha subunit of Gs promotes its removal from the plasma membrane.

As assessed both by cholera-toxin-catalysed ADP-ribosylation and by immunoblotting with an anti-peptide antiserum raised against the C-terminal decapeptide of forms of Gs alpha (the alpha subunit of the stimulatory guanine nucleotide-binding protein), rat glioma C6 BU1 cells express two forms of Gs alpha: a major 44 kDa form and a much less prevalent 42 kDa form. We examined the effects of guanine nucleotides on the interaction of the 44 kDa form with the plasma membrane. Incubation of membranes of C6 BU1 cells with poorly hydrolysed analogues of GTP, but not with analogues of either ATP or GDP, caused the release of this Gs alpha from the membrane fraction. Release of Gs alpha was observed within 5 min, and continued throughout the incubation period. After treatment with guanosine 5'-[beta gamma-imido]triphosphate for 60 min, some 75% of this polypeptide had been released from its site of membrane attachment. These experiments demonstrate that Gs alpha need not remain associated invariantly with the plasma membrane.

Active site of C3a anaphylatoxin: contributions of the lipophilic and orienting residues.

Activation of the serum complement cascade generates C3a anaphylatoxin, a primary mediator of inflammation. The active-site pentapeptide from the COOH terminus of C3a, Leu-Gly-Leu-Ala-Arg (residues 73-77), exhibits the inflammatory activities and specificity of the native 77-residue polypeptide. Functionally important features of this active site were studied by testing the ability of 22 synthetic analogues of this pentapeptide to contract isolated muscle strips from guinea pig ileum and to desensitize this tissue to contraction induced by human C3a or C5a. The C3a receptors on mast cells and basophils probably contain lipophilic groups that interact with the lipophilic side chains of Leu-73 and Leu-75 and charged groups that interact with the carboxylate and guanidinium groups of Arg-77. The lipophilic contribution of Leu-73 is modest and sterically nonspecific while that of Leu-75 is substantial and sterically specific. Gly-74 and Ala-76 appear to position and orient the adjacent residues Leu-73, Leu-75, and Arg-77 for optimal receptor binding. The contribution of Gly-74 is neither conformationally nor sterically specific while that of Ala-76 is both conformationally and sterically specific. The cellular C3a receptors evidently interact most efficiently with peptides ending in -Leu-Ala-Arg-OH.

Roles of aspartic acid 15 and 21 in glucagon action: receptor anchor and surrogates for aspartic acid 9.

The discovery of aspartic acid at position 9 in glucagon to be a critical residue for transduction has spurred renewed efforts to identify other strategic residues in the peptide sequence that dictate either receptor binding or biological activity. It also became apparent from further studies that Asp9 operates in conjunction with His1 in the activation mechanism that follows binding to the glucagon receptor. Indeed, it was later demonstrated that the protonatable histidine imidazole is important for transduction. It is likely that the interaction of a positively charged histidine 1 with a negatively charged aspartic acid 9 might be part of the triggering step at the molecular level. Two other aspartic acid residues in glucagon are capable of assuming a similar role, namely that of contributing to an electrostatic attraction with histidine via a negative carboxylate. These studies were conducted to investigate the role of aspartic acid 15 and 21 in glucagon action. Evidence reported here, gathered from 31 replacement analogs, supports the idea that in the absence of the requisite carboxyl group at position 9, histidine utilizes Asp21 or Asp15 as a compensatory site. Asp15 was also found to be indispensable for binding and may serve to tether the hormone to the receptor protein at the binding site. It is also demonstrated that these new findings promote the design of better glucagon antagonists.

Potentiation of glucose-induced insulin release in islets by desHis1[Glu9]glucagon amide.

Glucagon and secretin and some of their hybrid analogs potentiate glucose-induced release of insulin from isolated mouse pancreatic islets. It was recently shown that the synthetic glucagon analog, desHis1[Glu9]glucagon amide, does not stimulate the formation of cyclic adenosine monophosphate in the rat hepatocyte membrane, but binds well to the glucagon receptor and is a good competitive antagonist of glucagon. In the present study the effect of this analog on isolated islets was examined. desHis1-[Glu9]glucagon amide at 3 x 10(-7) M, in the presence of 0.01 M D-glucose, increased the release of insulin by 30% and maintained that level for the full 30-min test period. The rate of insulin release returned to the glucose-induced base line after removal of the peptide. The same insulin level was produced by 3 x 10(-9) M glucagon, and at 3 x 10(-7) M glucagon insulin release was enhanced 290% above the glucose base line.

Cholera toxin treatment produces down-regulation of the alpha-subunit of the stimulatory guanine-nucleotide-binding protein (Gs).

The effect of activation of the alpha-subunit(s) of the stimulatory guanine-nucleotide-binding protein, Gs, on levels of this polypeptide(s) associated with the plasma membrane of L6 skeletal myoblasts was ascertained. Incubation of these cells with cholera toxin led to a time- and concentration-dependent 'down-regulation' of both 44 and 42 kDa forms of Gs alpha as assessed by immunoblotting with an anti-peptide antiserum (CS1) able to identify the extreme C-terminus of Gs. The effect of cholera toxin was specific for Gs; levels of Gi alpha in membranes of cholera toxin-treated cells were not different from untreated cells. Down-regulation of Gs was absolutely dependent upon prior ADP-ribosylation, and hence activation of Gs and was not mimicked by other agents which elevate intracellular levels of cyclic AMP. Pretreatment with pertussis toxin, which catalyses ADP-ribosylation of Gi but not of Gs, did not down-regulate either Gi or Gs, demonstrating that covalent modification by ADP-ribosylation is alone not a signal for removal of G-proteins from the plasma membrane.

The role of histidine-1 in glucagon action.

The identification of position 9 aspartic acid in glucagon as a critical residue for transduction reinforced the notion that specific residues in the peptide sequence dictate either receptor recognition or biological activity. It was evident from our studies that Asp9 operates in conjunction with His1 as part of the activation mechanism that follows binding to the glucagon receptor. This investigation was conducted to delineate structural features of histidine that contribute to its role in glucagon action. We report, based on binding and activity data from 10 replacement analogs, that the imidazole ring of His1 furnishes an aromatic determinant for receptor binding affinity and that its protonatable imidazole nitrogen is important for transduction.

Selective stabilization of the high affinity binding conformation of glucagon receptor by the long splice variant of Galpha(s).

To analyze functional differences in the interactions of the glucagon receptor (GR) with the two predominant splice variants of Galpha(s), GR was covalently linked to the short and the long forms Galpha(s)-S and Galpha(s)-L to produce the fusion proteins GR-Galpha(s)-S and GR-Galpha(s)-L. GR-Galpha(s)-S bound glucagon with an affinity similar to that of GR, while GR-Galpha(s)-L showed a 10-fold higher affinity for glucagon. In the presence of GTPgammaS, GR-Galpha(s)-L reverted to the low affinity glucagon binding conformation. Both GR-Galpha(s)-L and GR-Galpha(s)-S were constitutively active, causing elevated basal levels of cAMP even in the absence of glucagon. A mutant GR that failed to activate G(s) (G23D1R) was fused to Galpha(s)-L. G23D1R-Galpha(s)-L bound glucagon with high affinity, but failed to elevate cAMP levels, suggesting that the mechanisms of GR-mediated Galpha(s)-L activation and Galpha(s)-L-induced high affinity glucagon binding are independent. Both GR-Galpha(s)-S and GR-Galpha(s)-L bound the antagonist desHis(1)[Nle(9),Ala(11),Ala(16)]glucagon amide with affinities similar to GR. The antagonist displayed partial agonist activity with GR-Galpha(s)-L, but not with GR-Galpha(s)-S. Therefore, the partial agonist activity of the antagonist observed in intact cells appears to be due to GRs coupled to Galpha(s)-L. We conclude that Galpha(s)-S and Galpha(s)-L interact differently with GR and that specific coupling of GR to Galpha(s)-L may account for GTP-sensitive high affinity glucagon binding.