Edward W. McBride

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.

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.

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.

Interspecies polymorphisms confer constitutive activity to the Mastomys cholecystokinin-B/gastrin receptor.

The enteroendocrine hormone, gastrin, exerts trophic effects on the gastric mucosa through the CCK-B/gastrin receptor (CCK-BR). To varying degrees in different species, excess circulating gastrin leads to proliferation of enterochromaffin-like cells and to the development of gastric carcinoid tumors. The African rodent, Mastomys natalensis, is distinguished from other mammals by its propensity toward CCK-BR-mediated growth even in the absence of hypergastrinemia. Here, we report that theMastomys CCK-BR, when expressed in COS-7 cells, differs from the respective human, canine, and rat receptor homologs by its ability to trigger ligand-independent (i.e., constitutive) inositol phosphate formation. To define the molecular basis of this observation, a series of Mastomys-human chimeric receptors was investigated. Functional characterization of these constructs revealed that a limited segment of the Mastomys CCK-BR, transmembrane domain VI through the C-terminal end, is sufficient to confer constitutive activity to the human protein. Mutagenesis studies within this CCK-BR region defined a combination of threeMastomys amino acids that, when introduced into the human receptor, together conferred a level of ligand-independent signaling comparable with the Mastomys CCK-BR. Complementing prior observations that single point mutations can lead to ligand-independent signaling, our findings suggest that multiple naturally occurring amino acid polymorphisms and/or mutations may together result in an enhanced basal level of receptor activity.

The CCK-B/gastrin receptor. Identification of amino acids that determine nonpeptide antagonist affinity.

Gastrin and cholecystokinin (CCK) share an identical carboxy-terminal pentapeptide amide, a domain critical for receptor binding. Based on the binding of agonists as well as antagonists, receptors within the gastrin/CCK family have traditionally been divided into two major subtypes: CCK-A and CCK-B/gastrin.l The CCK-A receptor is found in pancreas, gallbladder, and isolated brain nuclei. CCK-B/gastrin receptors are present on smooth muscle cells and parietal cells (“gastrin” receptors) and are the predominant brain CCK receptor (“CCK-B”). Although CCK-A and CCK-B/gastrin receptors can easily be distinguished on the basis of both agonist and antagonist binding, small differences in agonist binding have created controversy regarding the existence of distinct “CCK-B” and “gastrin” receptor subtypes. Our data suggest that the CCK-B and gastrin receptors represent a single subtype2 and that species differences underlie part of the heterogeneity in ligand binding reported within the CCK-B/gastrin receptor ~ubclass.~ As a first step towards understanding receptor function at a molecular level, we used a COS-7 cell expression cloning system to isolate a cDNA encoding the parietal cell gastrin receptor (FIG. 1). The first requirement in this cloning strategy was to isolate mRNA from cells with abundant gastrin receptors. On the basis of A. H. Soll’s earlier observations,4 we used an enriched (95%) preparation of canine parietal cells as starting material. From these cells, size selected (> 1.5 kb) mRNA was isolated and used to make a cDNA expression library in the vector pcDNAl (Invitrogen). Pools of library DNA, representing 3,000-10,000 primary recombinants, were transfected into COS-7 cells. Forty-eight hours after transfection, cells were incubated with IzI CCK-8 as well as with lSI D-Tyr-Gly-[(Nle28*31)-CCK-26-33], a CCK analog that includes a free amino group available for chemical cross-linking? Inasmuch as COS-7 cells do not normally have gastrin or CCK receptors, only cells expressing the recombinant gastrin receptor bound the radioligand. Following the binding assay, excess radioligand was washed away, and the cells were fixed in glutaraldehyde. Slides were then dipped in photoemulsion, exposed in the dark for 72 hours, developed, and examined under the microscope. After two million primary recombinants were screened, a pool expressing the

Point Mutations in Either Subunit of the GABAB Receptor Confer Constitutive Activity to the Heterodimer

The GABA receptor (GABABR) is a class C G protein-coupled receptor (GPCR) that functions as an obligate heterodimer, composed of two heptahelical subunits, GABABR subunit 1 (R1) and GABABR subunit 2 (R2). In this study, we generated and pharmacologically characterized constitutively active GABABR mutants as novel tools to explore the molecular mechanisms underlying receptor function. A single amino acid substitution, T290K, in the R1 agonist binding domain results in ligand-independent signaling when this mutant subunit is coexpressed with wild-type R2. Introduction of a Y690V mutation in the putative G protein-coupling domain of R2 is sufficient to confer moderate constitutive activity when this subunit is expressed alone. Activity of the Y690V mutant can be markedly enhanced with coexpression of wild-type R1. Coexpression of both mutant subunits (R1-T290K and R2-Y690K) leads to a further increase in basal signaling. Potencies of the full agonists R-(+)-β-(aminomethyl)-4-chlorobenzenepropanoic acid hydrochloride (baclofen) and GABA are increased at the constitutively active versus the corresponding wild-type receptors. The mutant GABABR variants provided a sensitive probe enabling detection of inverse or partial agonist activity of molecules previously considered neutral antagonists. Our studies using constitutively active isoforms provide independent support for a model of GABABR function that takes into account 1) ligand binding by R1, 2) signal transduction by R2, and 3) modulation of R2-induced function by R1. Furthermore, we demonstrate that certain hallmark features of constitutive activity as originally established with class A GPCRs (e.g., enhanced agonist potency and affinity), are more generally applicable, as suggested by our finding with a class C heterodimeric receptor.

Identification of a series of CCK-2 receptor nonpeptide agonists: Sensitivity to stereochemistry and a receptor point mutation

The search for small-molecule drugs that act at peptide hormone receptors has resulted in the identification of a wide variety of antagonists. In contrast, the discovery of nonpeptide agonists has been far more elusive. We have used a constitutively active mutant of the cholecystokinin 2 receptor (CCK-2R) as a sensitive screen to detect ligand activity. Functional assessment of structural analogs of the prototype CCK-2R antagonist, L-365,260 [3R-N- (2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl)-N′-(3-methylphenyl)urea], resulted in the identification of a series of agonists. Each of the active molecules is an S enantiomer, whereas the corresponding R stereoisomers have little or no activity. Further in vitro and in vivo assessment at the wild-type receptor indicated that efficacy of the two most active ligands approached that of the endogenous hormone. The function of selected R and S enantiomers was differentially sensitive to a point mutation, N353L, within the putative CCK-2R ligand pocket. The results of this study highlight the potential of constitutively active receptors as drug screening tools and the interdependence of ligand stereochemistry and receptor conformation in defining drug efficacy.

Conserved cholecystokinin receptor transmembrane domain IV amino acids confer peptide affinity

The Cholecystokinin type 1 and type 2 receptors (CCK-1R and CCK-2R) share >50% amino acid identity, as well as subnanomolar affinity for the endogenous peptide cholecystokinin octapeptide (CCK-8). Although it is likely that these two receptor subtypes share amino acids that confer CCK-8 affinity, it has been difficult to identify such residues. We have examined the role of several transmembrane domain (TMD) IV residues that are common to both CCK receptor subtypes. In both the CCK-1R and CCK-2R, we demonstrate that alanine substitution of two TMD IV residues, which are highly conserved among all known CCK receptor subtypes and species homologs, significantly decrease CCK-8 affinity. Despite the observed decrease in peptide binding, the mutant receptors maintain close to wild-type affinity for the respective subtype selective nonpeptide ligands, 3H-labeled L-364,714 (CCK-1R) and 3H-labeled L-365,260 (CCK-2R), suggesting conserved tertiary structure of these mutants. Assessment of CCK-8-induced inositol phosphate production at each of the mutant CCK receptors revealed normal peptide efficacy. In contrast, peptide potencies are reduced in parallel with the observed decreases in affinity. Taken together, these findings suggest that important peptide affinity determinants are localized on TMD IV, a region that has not previously been considered a major contributor to ligand affinity in either CCK receptors or other G protein-coupled peptide receptors.