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.

Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase.

We prepared a stable cell line expressing the glucagon receptor to characterize the effect of G(s)-coupled receptor stimulation on extracellular signal-regulated protein kinase 1/2 (ERK1/2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase/ERK kinase (MEK1/2) and ERK1/2. Inhibition of either PKA or MEK1/2 blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1/2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1/2 phosphorylation. We conclude that glucagon-induced MEK1/2 and ERK1/2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation.

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.

The effect of low dose micronized 17ss-estradiol on bone turnover, sex hormone levels, and side effects in older women: a randomized, double blind, placebo-controlled study.

The purpose of this study was to examine the effects of three doses (0.25, 0.5, and 1.0 mg/day) of micronized 17ss-estradiol on bone turnover, sex hormone levels, and side effects compared with placebo in healthy older women. The study design was randomized, double blind, and placebo controlled. The setting was a university clinical research center. Healthy, community-living women over 65 yr of age participated in the study. The main outcome measures were serum and urinary biochemical markers of bone resorption and formation at baseline, 6 and 12 weeks on treatment, and 6 and 12 weeks off treatment. Markers of bone resorption were N-telopeptides of type I collagen, C-telopeptides of type I collagen, and total deoxypyridinoline cross-links; formation markers were bone alkaline phosphatase, osteocalcin, and N-terminal procollagen peptides. We also measured serum estradiol, estrone, and sex hormone-binding globulin levels at baseline, 12 weeks on treatment, and 12 weeks posttreatment. All markers of bone resorption significantly decreased at 12 weeks on treatment compared with placebo and returned toward baseline at 12 weeks posttreatment. Two markers of bone formation, bone alkaline phosphatase and N-terminal procollagen peptides, significantly decreased 12 weeks posttreatment, but the decrease in osteocalcin varied with time and estrogen dose. Based on equivalence testing, the response of markers of bone turnover to therapy with 0.25 mg/day was similar to that seen with 1.0 mg/day. Serum estradiol increased compared with baseline in all treatment groups and compared with placebo in the two higher dose groups. Breast tenderness, bleeding, and endometrial changes were significantly less frequent in the 0.25 mg/day and placebo groups compared with the higher dose groups. We conclude that low dose of estrogen (0.25 mg/day 17ss-estradiol) reduced bone turnover to a similar degree as that seen with usual replacement therapy (1.0 mg/day 17ss-estradiol), but had a side effect profile similar to that of placebo. In our study additional increases in estradiol levels, as seen with 0.5 and 1.0 mg/day 17ss-estradiol treatment, resulted in more side effects without evidence of additional benefit to bone. These data suggest that 0.25 mg/day 17ss-estradiol may be an effective and tolerable agent for the treatment of osteoporosis in older women.

Two cytoplasmic loops of the glucagon receptor are required to elevate cAMP or intracellular calcium.

The glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) sharing little amino acid sequence homology with the larger rhodopsin-like GPCR family. To identify the components of the glucagon receptor necessary for G-protein coupling, we replaced sequentially all or part of each intracellular loop (i1, i2, and i3) and the C-terminal tail of the glucagon receptor with the 11 amino acids comprising the first intracellular loop of the D4 dopamine receptor. When expressed in transiently transfected COS-1 cells, the mutant receptors fell into two different groups with respect to hormone-mediated signaling. The first group included the loop i1 mutants, which bound glucagon and signaled normally. The second group comprised the loop i2 and i3 chimeras, which caused no detectable adenylyl cyclase activation in COS-1 cells. However, when expressed in HEK 293T cells, the loop i2 or i3 chimeras caused very small glucagon-mediated increases in cAMP levels and intracellular calcium concentrations, with EC50 values nearly 100-fold higher than those measured for wild-type receptor. Replacement of both loops i2 and i3 simultaneously was required to completely abolish G protein signaling as measured by both cAMP accumulation and calcium flux assays. These results show that the i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G proteins by rhodopsin-like GPCRs.

Positively charged residues at positions 12, 17, and 18 of glucagon ensure maximum biological potency.

Glucagon is a peptide hormone that plays a central role in the maintenance of normal circulating glucose levels. Structure-activity studies have previously demonstrated the importance of histidine at position 1 and the absolute requirement for aspartic acid at position 9 for transduction of the hormonal signal. Site-directed mutagenesis of the receptor protein identified Asp64 on the extracellular N-terminal tail to be crucial for the recognition function of the receptor. In addition, antibodies generated against aspartic acid-rich epitopes from the extracellular region competed effectively with glucagon for receptor sites, which suggested that negative charges may line the putative glucagon binding pocket in the receptor. These observations led to the idea that positively charged residues on the hormone may act as counterions to these sites. Based on these initial findings, we synthesized glucagon analogs in which basic residues at positions 12, 17, and 18 were replaced with neutral or acidic residues to examine the effect of altering the positive charge on those sites on binding and adenylyl cyclase activity. The results indicate that unlike N-terminal histidine, Lys12, Arg17, and Arg18 of glucagon have very large effects on receptor binding and transduction of the hormonal signal, although they are not absolutely critical. They contribute strongly to the stabilization of the binding interaction with the glucagon receptor that leads to maximum biological potency.

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.

Antibodies against specific extracellular epitopes of the glucagon receptor block glucagon binding.

Polyclonal antibodies were prepared against synthetic peptides corresponding to four different extramembrane segments of the rat glucagon receptor. The antibodies bound specifically to native glucagon receptor as judged by immunofluorescence microscopy of cultured cells expressing a synthetic gene for the receptor. Antibodies to peptides designated PR-15 and DK-12 were directed against amino acid residues 103-117 and 126-137, respectively, of the extracellular N-terminal tail. Antibody to peptide KD-14 was directed against residues 206-219 of the first extracellular loop, and antibody to peptide ST-18, against the intracellular C-terminal tail, residues 468-485. The DK-12 and KD-14 antibodies, but not the PR-15 and ST-18 antibodies, could effectively block binding of 125I-labeled glucagon to its receptor in liver membranes. Incubation of these antibodies with rat liver membranes resulted in both a decrease in the maximal hormonal binding capacity and an apparent decrease in glucagon affinity for its receptor. These effects were abolished in the presence of excess specific peptide antigen. In addition, DK-12 and KD-14 antibodies, but not PR-15 and ST-18 antibodies, interfered with glucagon-induced adenylyl cyclase activation in rat liver membranes and behaved as functional glucagon antagonists. These results demonstrate that DK-12 and KD-14 antibodies are pharmacologically active glucagon antagonists and strongly suggest that residues 126-137 of the N-terminal tail and residues 206-219 of the first extracellular loop contain determinants of ligand binding and may comprise the primary ligand-binding site on the glucagon receptor.

Characterization of deletion and truncation mutants of the rat glucagon receptor. Seven transmembrane segments are necessary for receptor transport to the plasma membrane and glucagon binding.

Glucagon receptor mutants were characterized with the aim of elucidating minimal structural requirements for proper biosynthesis, ligand binding, and adenylyl cyclase coupling. One N-terminal deletion mutant and five truncation mutants with progressively shorter C termini were expressed in transiently transfected monkey kidney (COS-1) cells. Each truncation mutant was designed so that the truncated C-terminal tail would remain on the cytoplasmic surface of the receptor. In order to characterize the cellular location of the expressed receptor mutants, a highly specific, high affinity antipeptide antibody was prepared against the extracellular, N-terminal tail of the receptor. Immunoblot analysis and immunofluorescence microscopy showed that the presence of all seven putative transmembrane segments, but not not an intact N-terminal tail, was required for cell surface expression of the receptor. Membranes from cells expressing receptor mutants lacking a large portion of the N-terminal tail or any of the seven putative transmembrane segments failed to bind glucagon. Membranes from cells expressing the C-terminal tail truncation mutants, which retained all seven transmembrane segments, bound glucagon with affinities similar to that of the native receptor and activated cellular adenylyl cyclase in response to glucagon. These results indicate that all seven helices are necessary for the proper folding and processing of the glucagon receptor. Glycosylation is not required for the receptor to reach the cell surface, and it may not be required for ligand binding. However, the N-terminal extracellular portion of the receptor is required for ligand binding. Most of the distal C-terminal tail is not necessary for ligand binding, and the absence of the tail may increase slightly the receptor binding affinity for glucagon. The C-terminal tail is also not necessary for adenylyl cyclase coupling and therefore does not play a direct role in G protein (GS) activation by the glucagon receptor.

Synthesis and expression of a gene for the rat glucagon receptor. Replacement of an aspartic acid in the extracellular domain prevents glucagon binding.

In order to facilitate structure-function studies of the glucagon receptor by site-directed mutagenesis, we have designed and synthesized a gene for the rat glucagon receptor. The gene codes for the native 485-amino-acid protein but contains 91 unique restriction sites. To characterize gene expression, a highly specific, high affinity antipeptide antibody was prepared against the receptor. The synthetic gene was expressed in transiently transfected monkey kidney (COS-1) cells. COS cells expressing the synthetic receptor gene bound glucagon with affinity and specificity similar to that of hepatocytes containing native receptor. The transfected COS cells also showed increased intracellular cAMP levels in response to glucagon. The functional role of an aspartic acid residue in the NH2-terminal tail of the receptor was tested by site-directed mutagenesis. This site in the related growth hormone releasing factor receptor was shown to be responsible for the little mouse (lit) genetic defect that results in mice of small size with hypoplastic pituitary glands. Mutant glucagon receptors with amino acid replacements of Asp64 were expressed at normal levels in COS cells but failed to bind glucagon. These results indicate that amino acid Asp64 may play a key role in glucagon binding to receptor.

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.

Multiple-site replacement analogs of glucagon. A molecular basis for antagonist design.

Extensive structure activity analysis has allowed us to identify specific residues in the glucagon sequence that are responsible for either receptor recognition or signal transduction. For instance, we have demonstrated that aspartic acid 9 and histidine 1 are essential for activation, and that an ionic interaction between the negative carboxylate and the protonated imidazole may contribute to the activation reaction at the molecular level. In the absence of the carboxylic group at position 9, aspartic 21 or aspartic 15 might furnish distal electrostatic effects to maintain partial agonism. Further investigation established that each of the 4 serine residues in the hormone play distinct roles. Serine 8 provides an important determinant of binding. Whereas neither serines 2, 11, nor 16 are required for receptor recognition. We have shown that serine 16 is essential for signal transduction and thus have identified it to be the third residue in glucagon to participate in a putative catalytic triad together with aspartic 9 and histidine 1, in the transduction of the glucagon response. In this work, we utilized insights into the functional significance of particular residues in the peptide appropriated from our structure-function assignments, as the basis of a molecular approach for the design of active-site directed antagonists of glucagon. The importance as well as the accuracy of our findings are confirmed by the synthesis of a series of improved glucagon antagonists based on replacements at positions 1, 9, 11, 16, and 21. The inhibition index, (I/A)50, of our best antagonist des-His1-[Nle9-Ala11-Ala16]glucagon amide, has been improved 10-fold over the previous best glucagon inhibitor.

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.

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.

The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the novel Gq family.

The thromboxane A2 (TXA2) receptor in human platelets is coupled to a pertussis toxin-insensitive G protein whose identity has remained unknown. Candidates for this role include the atypical G protein known as Gz and members of a recently discovered G protein family known as Gq. Because of the proven utility of antibodies directed against the C terminus of G protein alpha subunits as functional probes, we prepared an antibody against a synthetic decapeptide corresponding to the C-terminal sequence shared by alpha 11 and alpha q, two members of the new family. This antibody (QL) does not recognize known alpha subunits but selectively binds to a 42-kDa protein in a variety of tissues, including human platelet membranes. QL and two other C-terminal antibodies, QN and AS, known to recognize alpha z and alpha i2, respectively, were tested for their ability to block agonist-stimulated GTPase activity in human platelet membranes. Pretreatment of platelet membranes with AS has previously been shown to interfere with alpha 2 adrenergic receptor-mediated inhibition of adenylylcyclase. As expected, only AS antibody produced inhibition of alpha 2 receptor-stimulated GTPase. Pretreatment of membranes with QL, but not QN or AS, caused marked inhibition of TXA2 receptor-stimulated GTPase. This identifies the G protein coupled to human platelet TXA2 receptors as a member of the novel Gq family.

G-protein beta gamma dimers. Membrane targeting requires subunit coexpression and intact gamma C-A-A-X domain.

Attachment of heterotrimeric G-proteins to the inner face of the plasma membrane is fundamental to their role as signal transducers by allowing interaction with both receptors and effectors. Certain G-protein alpha subunits are anchored to the membrane by covalent myristoylation. The beta gamma complex is required for G-protein interaction with receptors and is independently membrane associated through an unknown mechanism. A series of carboxyl-terminal modifications including isoprenylation which may contribute to membrane attachment has been identified recently in G-protein gamma subunits. Expression and membrane targeting of beta and gamma subunits were examined in COS cells. The expression of either subunit was found to require cotransfection with both beta and gamma cDNAs. Mutation of the carboxyl-terminal cysteine residue of gamma shown to undergo isoprenylation and carboxymethyl-esterification preserved beta gamma expression but blocked isoprenylation and membrane attachment. These results implicate the carboxyl-terminal processing of G-protein gamma subunits and beta coexpression as necessary and sufficient for membrane targeting of the beta gamma complex.

Position 9 replacement analogs of glucagon uncouple biological activity and receptor binding.

Recent studies on the glucagon antagonist des-His1-[Glu9]glucagon amide have resulted in pure inhibitors of the hormone, suggesting that the inhibitory properties may be centered around position 9. The present study was designed to investigate the chemical characteristics of substitutions in position 9 of glucagon that determine binding affinity and biological activity. Twenty replacement analogs of position 9 of glucagon were synthesized and assessed for their ability to bind to the glucagon receptor in rat hepatocyte membranes and to activate adenylate cyclase. Any substitution of aspartic acid 9 was accompanied by a severely diminished capacity to transmit the biological signal, while retaining receptor binding affinity. These results are an indication of an uncoupling of receptor binding and biological activity at this locus and define a central role of aspartic acid 9 in glucagon activity. Single replacement or deletion of either His1 or Asp9 in glucagon caused a 20- to 50-fold decrease in cyclase activity, whereas these same changes made in tandem caused virtually complete loss of activity, with decreases of 10(4)-to 10(6)-fold. These observations have led us to speculate that, at the molecular level, the region of glucagon required for transduction of the biological response may be distinct from the binding region and is mediated by a coupled interaction between His1 and Asp9 of the hormone and a complementary functional site of the glucagon receptor.

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.