Cecilia G. Unson

Receptor and effector interactions of Gs Functional studies with antibodies to the αs carboxyl‐terminal decapeptide

Antibodies generated to a synthetic decapeptide, RMHLRQYELL, representing the carboxyl‐terminus of Gs‐α 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[γ]S‐ or fluoride‐preactivated bovine brain membranes; thus binding of αs 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 electors in the membrane.

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.

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 Gs-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.

Glucagon-Expressing Neurons within the Retina Regulate the Proliferation of Neural Progenitors in the Circumferential Marginal Zone of the Avian Eye

Glucagon-expressing retinal amacrine cells have been implicated in regulating postnatal ocular growth. Furthermore, experimentally accelerated rates of ocular growth increase the number of neurons added to the peripheral edge of the retina. Accordingly, we assayed whether glucagon-expressing neurons within the retina regulate the proliferation of progenitors in the circumferential marginal zone (CMZ) of the postnatal chicken eye. We found that glucagon-containing neurites are heavily clustered within the CMZ at the peripheral edge of the retina. Many of these neurites originate from a cell type that is distinct from other types of retinal neurons, which we termed large glucagon-expressing neurons (LGENs). The LGENs are immunoreactive for glucagon and glucagon-like peptide 1 (GLP1), have a unipolar morphology, produce an axon that projects into the CMZ, and are found only in ventral regions of the retina. In dorsal regions of the retina, a smaller version of the LGENs densely ramifies neurites in the CMZ. Intraocular injections of glucagon or GLP1 suppressed the proliferation of progenitors in the CMZ, whereas a glucagon-receptor antagonist promoted proliferation. In addition, we found that glucagon, GLP1, and glucagon antagonist influenced the number of progenitors in the CMZ. We conclude that the LGENs may convey visual information to the CMZ to control the addition of new cells to the edge of the retina. We propose that glucagon/GLP1 released from LGENs acts in opposition to insulin (or insulin-like growth factor) to regulate precisely the proliferation of retinal progenitors in the CMZ.

Detection of the major pertussis toxin substrate of human leukocytes with antisera raised against synthetic peptides

Antisera raised against the carboxy‐terminal decapeptide (KENLKDCGLF) of transducin‐α 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.

GTP-Binding proteins in brain and neutrophil are tethered to the plasma membrane via their amino termini

Using specific antisera raised against synthetic peptides, we find that three distinct GTP-binding protein alpha subunits remain bound to the plasma membrane even after activation with nonhydrolyzable GTP analog. Trypsin cleaves each alpha subunit at a site near the amino-terminus, and quantitatively releases the large fragment (comprising all but an amino-terminal 2 kDa piece) from the membrane. Our results indicate that alpha subunits are essentially cytoplasmic proteins tethered to the inner surface of the membrane via an amino terminal stalk.

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.

Glucagon-mediated internalization of serine-phosphorylated glucagon receptor and Gsα in rat liver

To assess glucagon receptor compartmentalization and signal transduction in liver parenchyma, we have studied the functional relationship between glucagon receptor endocytosis, phosphorylation and coupling to the adenylate cyclase system. Following administration of a saturating dose of glucagon to rats, a rapid internalization of glucagon receptor was observed coincident with its serine phosphorylation both at the plasma membrane and within endosomes. Co‐incident with glucagon receptor endocytosis, a massive internalization of both the 45‐ and 47‐kDa Gsα proteins was also observed. In contrast, no change in the subcellular distribution of adenylate cyclase or β‐arrestin 1 and 2 was observed. In response to des‐His1‐[Glu9]glucagon amide, a glucagon receptor antagonist, the extent and rate of glucagon receptor endocytosis and Gsα shift were markedly reduced compared with wild‐type glucagon. However, while the glucagon analog exhibited a wild‐type affinity for endosomal acidic glucagonase activity and was processed at low pH with similar kinetics and rates, its proteolysis at neutral pH was 3‐fold lower. In response to tetraiodoglucagon, a glucagon receptor agonist of enhanced biological potency, glucagon receptor endocytosis and Gsα shift were of higher magnitude and of longer duration, and a marked and prolonged activation of adenylate cyclase both at the plasma membrane and in endosomes was observed. The subsequent post‐endosomal fate of internalized Gsα was evaluated in a cell‐free rat liver endosome–lysosome fusion system following glucagon injection. A sustained endo‐lysosomal transfer of the two 45‐ and 47‐kDa Gsα isoforms was observed. Therefore, these results reveal that within hepatic target cells and consequent to glucagon‐mediated internalization of the serine‐phosphorylated glucagon receptor and the Gsα protein, extended signal transduction may occur in vivo at the locus of the endo‐lysosomal apparatus.

Differential expression of low molecular weight form of Gs-α 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.

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.