G Protein-coupled Receptors I. DIVERSITY OF RECEPTOR-LIGAND INTERACTIONS

G protein-coupled, seven-transmembrane segment receptors (GPCRs or 7TM receptors), with more than 1000 different members, comprise the largest superfamily of proteins in the body. Since the cloning of the first receptors more than a decade ago, extensive experimental work has uncovered multiple aspects of their function and challenged many traditional paradigms. However, it is only recently that we are beginning to gain insight into some of the most fundamental questions in the molecular function of this class of receptors. How can, for example, so many chemically diverse hormones, neurotransmitters, and other signaling molecules activate receptors believed to share a similar overall tertiary structure? What is the nature of the physical changes linking agonist binding to receptor activation and subsequent transduction of the signal to the associated G protein on the cytoplasmic side of the membrane and to other putative signaling pathways? The goal of the present review is to specifically address these questions as well as to depict the current awareness about GPCR structure-function relationships in general. (Endocrine Reviews 21: 90 ‐113, 2000)

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Uncovering Molecular Mechanisms Involved in Activation of G Protein-Coupled Receptors

GABA (γ-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian central nervous system, where it exerts its effects through ionotropic (GABAA/C) receptors to produce fast synaptic inhibition and metabotropic (GABAB) receptors to produce slow, prolonged inhibitory signals. The gene encoding a GABAB receptor (GABABR1) has been cloned1; however, when expressed in mammalian cells this receptor is retained as an immature glycoprotein on intracellular membranes2 and exhibits low affinity for agonists compared with the endogenous receptor on brain membranes. Here we report the cloning of a complementary DNA encoding a new subtype of the GABAB receptor (GABABR2), which we identified by mining expressed-sequence-tag databases. Yeast two-hybrid screening showed that this new GABABR2-receptor subtype forms heterodimers with GABABR1 through an interaction at their intracellular carboxy-terminal tails. Upon expression with GABABR2 in HEK293T cells, GABABR1 is terminally glycosylated and expressed at the cell surface. Co-expression of the two receptors produces a fully functional GABAB receptor at the cell surface; this receptor binds GABA with a high affinity equivalent to that of the endogenous brain receptor. These results indicate that, in vivo, functional brain GABAB receptors may be heterodimers composed of GABABR1 and GABABR2.

Heterodimerization is required for the formation of a functional GABAB receptor

Peptides of the glucagon/vasoactive-intestinal-peptide (VIP) peptide family share a considerable sequence similarity at their N-terminus. They either start with Tyr-Ala, His-Ala or His-Ser which might be in part potential targets for dipeptidyl-peptidase IV, a highly specialized aminopeptidase removing dipeptides only from peptides with N-terminal penultimate proline or alanine. Growth-hormone-releasing factor (1-29)amide and gastric inhibitory peptide/glucose-dependent insulinotropic peptide (GIP) with terminal Tyr-Ala as well as glucagon-like peptide-1(7-36)amide/insulinotropin [GLP-1(7-36)amide] and peptide histidine methionine (PHM) with terminal His-Ala were hydrolysed to their des-Xaa-Ala derivatives by dipeptidyl-peptidase IV purified from human placenta. VIP with terminal His-Ser was not significantly degraded by the peptidase. The kinetics of the hydrolysis of GIP, GLP-1(7-36)amide and PHM were analyzed in detail. For these peptides Km values of 4-34 microM and Vmax values of 0.6-3.8 mumol.min-1.mg protein-1 were determined for the purified peptidase which should allow their enzymic degradation also at physiological, nanomolar concentrations. When human serum was incubated with GIP or GLP-1(7-36)amide the same fragments as with the purified dipeptidyl-peptidase IV, namely the des-Xaa-Ala peptides and Tyr-Ala in the case of GIP or His-Ala in the case of GLP-1(7-36)amide, were identified as the main degradation products of these peptide hormones. Incorporation of inhibitors specific for dipeptidyl-peptidase IV, 1 mM Lys-pyrrolidide or 0.1 mM diprotin A (Ile-Pro-Ile), completely abolished the production of these fragments by serum. It is concluded that dipeptidyl-peptidase IV initiates the metabolism of GIP and GLP-1(7-36)amide in human serum. Since an intact N-terminus is obligate for the biological activity of the members of the glucagon/VIP peptide family [e. g. GIP(3-42) is known to be inactive to release insulin in the presence of glucose as does intact GIP], dipeptidyl-peptidase-IV action inactivates these peptide hormones. The relevance of this finding for their inactivation and their determination by immunoassays is discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1993.tb17986.x

Dipeptidyl‐peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon‐like peptide‐1(7–36)amide, peptide histidine methionine and is responsible for their degradation in human serum

BACKGROUNDEndothelium-dependent vasodilation is abnormal in experimental models of diabetes mellitus. We postulated that abnormalities of endothelial function are also present in patients with insulin-dependent diabetes mellitus and may contribute to the pathogenesis of vascular disease in these individuals.METHODS AND RESULTSVascular reactivity was measured in the forearm resistance vessels of 15 patients with insulin-dependent diabetes mellitus and 16 age-matched normal subjects. No patient had hypertension or dyslipidemia. Each subject was pretreated with aspirin to inhibit endogenous production of prostanoids. Methacholine chloride (0.3 to 10 micrograms/min) was administered via the brachial artery to assess endothelium-dependent vasodilation. Sodium nitroprusside (0.3 to 10 micrograms/min) and verapamil (10 to 300 micrograms/min) were infused intra-arterially to assess endothelium-independent vasodilation; phenylephrine (0.3 to 3 micrograms/min) was administered to examine vasoconstrictor responsiven...

Impaired endothelium-dependent vasodilation in patients with insulin-dependent diabetes mellitus.

Many cell-surface receptors for hormones appear to exert their effects on target cells by interacting with specific guanine nucleotide binding regulatory proteins (G-proteins) which couple receptors to their second-messenger signal generation systems. A common intracellular second messenger, which is used by many hormones, is cyclic AMP. This is produced by adenylate cyclase, whose activity is controlled by two G-proteins, Gs which mediates stimulatory effects and Gi inhibitory effects on adenylate cyclase activity1. In liver, the hormone glucagon increases intracellular cAMP concentrations by activating adenylate cyclase by a Gs-mediated process. This effect of glucagon is antagonised by the hormone insulin, although the molecular mechanism by which insulin elicits its actions is obscure. However, insulin receptors exhibit a tyrosyl kinase activity2 and appear to interact with G-proteins2,3, perhaps by causing phosphorylation of them4. In type I diabetes, circulating insulin levels are abnormally low, giving rise to gross perturbations of metabolism as well as to a variety of complications such as ionic disturbances, neuropathies of the nervous system, respiratory and cardiovascular aberrations and predispostion to infection5. We show here that experimentally-induced type I diabetes leads to the loss of expression of G{ in rat liver. As it has been suggested that Gi may couple receptors to K+-channels6,7 as well as mediating the inhibition of adenylate cyclase, aberrations in the control of expression of this key regulatory protein in type I diabetes may be expected to lead to pleiotropic effects.

Abolition of the expression of inhibitory guanine nucleotide regulatory protein Gi activity in diabetes.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2889%2980622-2

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

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

Several glucagon analogs were synthesized in an effort to find derivatives that would bind with high affinity to the glucagon receptor of rat liver membranes but would not activate membrane-bound adenylate cyclase and, therefore, would serve as antagonists of the hormone. Measurements on a series of glucagon/secretin hybrids indicated that replacement of Asp9 in glucagon by Glu9, found in secretin, was the important sequence difference in the N terminus of the two hormones. Further deletion of His1 and introduction of a C-terminal amide resulted in des-His1-[Glu9]glucagon amide, which had a 40% binding affinity relative to that of native glucagon but caused no detectable adenylate cyclase activation in the rat liver membrane. This antagonist completely inhibited the effect of a concentration of glucagon that alone gave a full agonist response. It had an inhibition index of 12. The pA2 was 7.2. An attempt was made to relate conformation with receptor binding. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C18-silica columns.

https://www.pnas.org/content/pnas/84/12/4083.full.pdf

Synthetic peptide antagonists of glucagon.

https://www.jbc.org/content/266/5/2763.full.pdf

Cecilia G. Unson

Papers

Abolition of the expression of inhibitory guanine nucleotide regulatory protein Gi activity in diabetes.

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

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

Synthetic peptide antagonists of glucagon.

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