Martin Beinborn

A human glucagon-like peptide-1 receptor polymorphism results in reduced agonist responsiveness.

Glucagon-like peptide-1 (GLP-1) and its cognate receptor play an important physiological role in maintaining blood glucose homeostasis. A GLP-1 receptor (GLP-1R) polymorphism in which threonine 149 is substituted with a methionine residue has been recently identified in a patient with type 2 diabetes but was not found in non-diabetic control subjects. We have functionally assessed the recombinant GLP-1R variant after transient expression in COS-7 and HEK 293 cells. Compared to the wild type receptor, the variant GLP-1R showed (i) similar expression levels, (ii) 60-and 5-fold reduced binding affinities, respectively, for two GLP-1R full agonists, GLP-1 and exendin-4, and (iii) markedly decreased potencies of these peptides in triggering cAMP-mediated signaling (despite conserved efficacies). In contrast to full agonists, the efficacy of the primary GLP-1 metabolite/GLP-1R partial agonist, GLP-1 (9-36) amide, was essentially abolished by the T149M substitution. By hydropathy analysis, the polymorphism localizes to transmembrane domain 1, suggesting this receptor segment as a novel determinant of agonist affinity/efficacy. These findings reveal that naturally occurring sequence variability of the GLP-1R within the human population can result in substantial loss-of-function. A genetic link between the T149M variant and increased susceptibility to type 2 diabetes remains to be established.

The cholecystokinin-A receptor mediates inhibition of food intake yet is not essential for the maintenance of body weight

Food intake and body weight are determined by a complex interaction of regulatory pathways. To elucidate the contribution of the endogenous peptide cholecystokinin, mice lacking functional cholecystokinin-A receptors were generated by targeted gene disruption. To explore the role of the cholecystokinin-A receptor in mediating satiety, food intake of cholecystokinin-A receptor-/- mice was compared with the corresponding intakes of wild-type animals and mice lacking the other known cholecystokinin receptor subtype, cholecystokinin-B/gastrin. Intraperitoneal administration of cholecystokinin failed to decrease food intake in mice lacking cholecystokinin-A receptors. In contrast, cholecystokinin diminished food intake by up to 90% in wild-type and cholecystokinin-B/gastrin receptor-/- mice. Together, these findings indicate that cholecystokinin-induced inhibition of food intake is mediated by the cholecystokinin-A receptor. To explore the long-term consequences of either cholecystokinin-A or cholecystokinin-B/gastrin receptor absence, body weight as a function of age was compared between freely fed wild-type and mutant animals. Both cholecystokinin-A and cholecystokinin-B/gastrin receptor-/- mice maintained normal body weight well into adult life. In addition, each of the two receptor-/- strains had normal pancreatic morphology and were normoglycemic. Our results suggest that although cholecystokinin plays a role in the short-term inhibition of food intake, this pathway is not essential for the long-term maintenance of body weight.

Abnormal gastric histology and decreased acid production in cholecystokinin-B/gastrin receptor-deficient mice

BACKGROUND & AIMS The cholecystokinin (CCK)-B/gastrin receptor is one of several regulators of gastric acid secretion and mucosal growth. To elucidate the contribution of this receptor relative to other trophic and secretory factors, mice that lack the CCK-B/gastrin receptor have been generated and studied. METHODS Both alleles of the CCK-B/gastrin receptor were inactivated by targeted gene disruption. Analysis of the mice included measurement of basal gastric pH and plasma gastrin levels. In addition, multiple gastric mucosal cell types were identified by immunostaining and quantified. RESULTS Homozygous mutant mice were viable, fertile, and appeared grossly normal into adulthood. The receptor-deficient mice exhibited a marked increase in basal gastric pH (from 3.2 to 5.2) and an approximately 10-fold elevation in plasma gastrin concentration compared with wild-type controls. In the stomach of mutant animals, parietal and enterochromaffin-like cells were decreased, providing a likely explanation for the reduction in acid output. In the antrum, a decrease in somatostatin cell density and an increase in the gastrin cell number were observed, consistent with the concomitant elevation in circulating gastrin. CONCLUSIONS Together, these findings demonstrate the importance of the CCK-B/gastrin receptor in maintaining the normal cellular composition and function of the gastric mucosa.

A Drosophila dopamine 2-like receptor: Molecular characterization and identification of multiple alternatively spliced variants

Dopamine is an important neurotransmitter in the central nervous system of both Drosophila and mammals. Despite the evolutionary distance, functional parallels exist between the fly and mammalian dopaminergic systems, with both playing roles in modulating locomotor activity, sexual function, and the response to drugs of abuse. In mammals, dopamine exerts its effects through either dopamine 1-like (D1-like) or D2-like G protein-coupled receptors. Although pharmacologic data suggest the presence of both receptor subtypes in insects, only cDNAs encoding D1-like proteins have been isolated previously. Here we report the cloning and characterization of a newly discovered Drosophila dopamine receptor. Sequence analysis reveals that this putative protein shares highest homology with known mammalian dopamine 2-like receptors. Eight isoforms of the Drosophila D2-like receptor (DD2R) transcript have been identified, each the result of alternative splicing. The encoded heptahelical receptors range in size from 461 to 606 aa, with variability in the length and sequence of the third intracellular loop. Pharmacologic assessment of three DD2R isoforms, DD2R-606, DD2R-506, and DD2R-461, revealed that among the endogenous biogenic amines, dopamine is most potent at each receptor. As established for mammalian D2-like receptors, stimulation of the Drosophila homologs with dopamine triggers pertussis toxin-sensitive Gi/o-mediated signaling. The D2-like receptor agonist, bromocriptine, has nanomolar potency at DD2R-606, -506, and -461, whereas multiple D2-like receptor antagonists (as established with mammalian receptors) have markedly reduced if any affinity when assessed at the fly receptor isoforms. The isolation of cDNAs encoding Drosophila D2-like receptors extends the range of apparent parallels between the dopaminergic system in flies and mammals. Pharmacologic and genetic manipulation of the DD2Rs will provide the opportunity to better define the physiologic role of these proteins in vivo and further explore the utility of invertebrates as a model system for understanding dopaminergic function in higher organisms.

Four Missense Mutations in the Ghrelin Receptor Result in Distinct Pharmacological Abnormalities

The growth hormone secretagogue receptor (GHSR) plays an important role in regulating food intake and energy homeostasis. In this study, we compared the pharmacological properties of four reported variants of the human GHSR (I134T, V160M, A204E, and F279L) with those of the wild-type receptor. Corresponding recombinant receptors were transiently expressed in either human embryonic kidney 293 or COS-7 cells. Basal as well as ligand-induced signaling was assessed by luciferase reporter gene assays and measurement of inositol phosphate production. In addition, receptor expression levels were monitored by whole-cell enzyme-linked immunosorbent assay. Ligand-independent signaling of the wild-type GHSR is significantly reduced with introduction of either the V160M or F279L substitutions, whereas basal activity of the A204E mutant is not detectable. Ghrelin potency is markedly increased at the V160M mutant, whereas the I134T variant is unresponsive to this endogenous agonist. In contrast, the I134T mutant responds to a known GHSR inverse agonist, [d-Arg1, d-Phe5, d-Trp7,9, Leu11]-substance P (SP-analog), albeit with reduced efficacy. Activity of the SP-analog at the V160M and F279L mutants is comparable to the wild type (WT) value. The overall expression level of each of the four GHSR variants is reduced relative to WT; however, the ratio between the intracellular and plasma membrane receptor density remains comparable. Treatment with the SP-analog significantly increases cell surface expression of each receptor with the exception of the A204E variant. Taken together, our studies reveal that naturally occurring GHSR mutations affect a wide range of pharmacologic properties. The physiological impact of these alterations within selected populations (e.g., obese, lean individuals) as well as the pharmacogenomic consequences of corresponding mutations remain to be further investigated.

Membrane-tethered ligands are effective probes for exploring class B1 G protein-coupled receptor function.

Class B1 (secretin family) G protein-coupled receptors (GPCRs) modulate a wide range of physiological functions, including glucose homeostasis, feeding behavior, fat deposition, bone remodeling, and vascular contractility. Endogenous peptide ligands for these GPCRs are of intermediate length (27–44 aa) and include receptor affinity (C-terminal) as well as receptor activation (N-terminal) domains. We have developed a technology in which a peptide ligand tethered to the cell membrane selectively modulates corresponding class B1 GPCR-mediated signaling. The engineered cDNA constructs encode a single protein composed of (i) a transmembrane domain (TMD) with an intracellular C terminus, (ii) a poly(asparagine-glycine) linker extending from the TMD into the extracellular space, and (iii) a class B1 receptor ligand positioned at the N terminus. We demonstrate that membrane-tethered peptides, like corresponding soluble ligands, trigger dose-dependent receptor activation. The broad applicability of this approach is illustrated by experiments using tethered versions of 7 mammalian endogenous class B1 GPCR agonists. In parallel, we carried out mutational studies focused primarily on incretin ligands of the glucagon-like peptide-1 receptor. These experiments suggest that tethered ligand activity is conferred in large part by the N-terminal domain of the peptide hormone. Follow-up studies revealed that interconversion of tethered agonists and antagonists can be achieved with the introduction of selected point mutations. Such complementary receptor modulators provide important new tools for probing receptor structure–function relationships as well as for future studies aimed at dissecting the tissue-specific biological role of a GPCR in vivo (e.g., in the brain vs. in the periphery).

Pharmacological Characterization of Human Incretin Receptor Missense Variants

Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gut-derived incretin hormones that regulate blood glucose levels. In addition to their widely accepted insulinotropic role, there is evidence that GLP-1 modulates feeding behavior and GIP regulates lipid metabolism, thereby promoting postprandial fat deposition. In this study, we investigated whether naturally occurring polymorphisms in the GLP-1 receptor (GLP-1R) and the GIP receptor (GIP-R) affect the pharmacological properties of these proteins. After transient expression of the receptors in human embryonic kidney 293 cells, basal and ligand-induced cAMP production were assessed by use of luciferase reporter gene assays. Our data reveal that the wild-type GIP-R displays a considerable degree of ligand-independent activity. In comparison, the GIP-R variants C46S, G198C, R316L, and E354Q show a marked decrease in basal signaling that may, at least in part, be explained by reduced cell surface expression. When stimulated with GIP, the C46S and R316L mutants display significantly reduced potency (>1000 and 25- fold, respectively) compared with wild type. Complementary competition binding assays further demonstrate that the C46S variant fails to bind radio-iodinated GIP, whereas all other GIP-R mutants maintain normal ligand affinity. In contrast to the GIP-R, the wild-type GLP-1R lacks constitutive activity. Furthermore, none of the 10 GLP-1R missense mutations showed an alteration in pharmacological properties versus wild type. The extent to which abnormalities in GIP-R function may lead to physiological changes or affect drug sensitivity in selected populations (e.g., obese, diabetic individuals) remains to be further investigated.

Search for α-helical propensity in the receptor-bound conformation of glucagon-like peptide-1

To elucidate the receptor-bound conformation of glucagon-like peptide-1 (GLP-1), a series of conformationally constrained GLP-1 analogues were synthesized by introducing lactam bridges between Lys(i) and Glu(i)(+4) to form alpha-helices at various positions. The activity and affinity of these analogues to GLP-1 receptors suggested that the receptor-bound conformation comprises two alpha-helical segments between residues 11-21 and 23-34. It is notable that the N-terminal alpha-helix is extended to Thr(11), and that Gly(22) plays a pivotal role in arranging the two alpha-helices. Based on these findings, a highly potent bicyclic GLP-1 analogue was synthesized which is the most conformationally constrained GLP-1 analogue reported to date.

CCK receptor polymorphisms: an illustration of emerging themes in pharmacogenomics

Polymorphisms in G-protein-coupled receptors can alter drug affinity and/or activity. In addition, genetic differences in amino acid sequences can induce ligand-independent signaling, which in turn can lead to disease. With growing efforts in the field of pharmacogenomics, it is anticipated that polymorphism-induced alterations in drug and/or receptor function will be a focus of increasing concern during the course of future drug-development efforts. In this review, the spectrum of pharmacological consequences that result from polymorphisms in the cholecystokinin CCK2 receptor will be discussed, thereby illustrating emerging themes in pharmacogenomics.

Influence of Selective Fluorination on the Biological Activity and Proteolytic Stability of Glucagon-like Peptide-1

The relative simplicity and high specificity of peptide therapeutics has fueled recent interest. However, peptide and protein drugs generally require injection and suffer from low metabolic stability. We report here the design, synthesis, and characterization of fluorinated analogues of the gut hormone peptide, GLP-1. Overall, fluorinated GLP-1 analogues displayed higher proteolytic stability with simultaneous retention of biological activity (efficacy). Fluorinated amino acids are useful for engineering peptide drug candidates and probing ligand-receptor interactions.