Biomedical subjects

Martin Beinborn

Publications and source records attributed to Martin Beinborn.

12 recordsLinked to original sources

PubMed2007

Identification of amino acid determinants of dopamine 2 receptor synthetic agonist function.

The human dopamine 2 receptor (hD2R) modulates locomotor activity, hormone secretion, and neuropsychiatric function. Current knowledge of the hD2R structure is in large part derived from mutagenesis studies and molecular pharmacologic analysis together with homology modeling using bovine rhodopsin as a template. In this study, we utilized comparison of the Drosophila D2-like receptor (DD2R) with the hD2R as a novel approach for identifying candidate amino acids that are determinants of ligand potency and/or efficacy. We focused our studies on four dopaminergic ligands that are used in the treatment of Parkinson's disease: bromocriptine, pergolide, piribedil, and ropinirole. All four ligands are potent agonists at the wild-type hD2R, whereas only bromocriptine shows comparable function at the DD2R. We performed site-directed mutagenesis to replace hD2R amino acids (modeled to project into the ligand binding pocket) with corresponding fly residues, and vice versa. Substitution of three amino acids in the hD2R with the homologous DD2R residues (V91A, C118S, and L170I) led to a pronounced loss of pergolide potency and efficacy. A converse triple amino acid substitution of human residues into the fly receptor (DD2R-A133V/S160C/I211L) markedly enhanced pergolide efficacy and potency at the mutant DD2R. The same substitutions also converted piribedil and ropinirole, which lacked appreciable activity on the DD2R, to partial agonists. These findings show the important role of these three residues in drug-receptor interactions. Our study illustrates that comparison of a mammalian receptor with an invertebrate homolog complements previously described strategies for defining G protein-coupled receptor structure-function relationships.

Amino Acid Sequence↗

PubMed2006

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-(+)-beta-(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.

Amino Acid Substitution↗

PubMed2006

Class B GPCRs: a hidden agonist within?

Class B G protein-coupled receptors (GPCRs) regulate a wide range of endocrine and neuroendocrine functions and are endogenously stimulated by moderately large peptide hormones. Current evidence suggests that the carboxyl termini of cognate peptides bind to the amino terminus of their G protein-coupled receptors (GPCRs) and that the peptides' amino terminal segments then dock to the heptahelical receptor portion to induce signaling. In this issue of Molecular Pharmacology, Dong et al. (p. 206) propose an alternative model of ligand-induced class B GPCR activation. Based primarily on studies with the secretin receptor, a prototype class B family member, they provide evidence that the endogenous peptide hormone does not function as an activator per se. Instead, this hormone (secretin) exposes a hidden, built-in agonist epitope that is present within the amino terminus of its target GPCR. Isolated oligopeptide fragments containing this epitope act as full agonists on the secretin receptor despite their lack of amino acid homology with the secretin hormone. These nonconventional agonists can be minimized to tripeptide molecules and still maintain biological activity. The study to be discussed introduces a novel paradigm of class B GPCR function, and may facilitate the elusive goal of finding small molecule agonist drugs for this therapeutically attractive group of receptors.

Animals↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

Basal receptor activation by locally produced glucagon-like peptide-1 contributes to maintaining beta-cell function.

Glucagon-like peptide 1 (GLP-1) is a physiological stimulus of pancreatic beta-cell function. This enteroendocrine hormone is produced by intestinal L cells, and is delivered via the bloodstream to GLP-1 receptors (GLP-1Rs) on pancreatic beta-cells. In addition, there is evidence that beta-cell GLP-1Rs maintain sustained basal activity even in the absence of intestinal peptide, an observation that has raised the question whether these receptors have some degree of ligand-independent function. Here, we provide an alternative explanation for basal receptor activity based on our finding that biologically relevant amounts of fully processed GLP-1 are locally generated by insulinoma cell lines, as well as by alpha-cells of isolated rat islets in primary culture. Presence of GLP-1 was established by immunocytochemistry, as well as by selective ELISAs and bioassays of cell supernatants. A GLP-1R antagonist significantly reduced insulin secretion/production in beta-TC-6 insulinoma cells and isolated rat islets, suggesting a functionally important loop between locally produced GLP-1 and its cognate receptor. Treatment with this antagonist also inhibited the growth of beta-TC-6 cells. These observations provide novel insight into the function of insulin-producing cell lines and native beta-cells during in vitro culture, and they support the idea that locally produced GLP-1 may play a role in intra-islet regulation.

Animals↗

PubMed2005

Ménétrier's disease in a patient with Helicobacter pylori infection is linked to elevated glucagon-like peptide-2 activity.

This report focuses on a 59-year-old male Japanese patient with Ménétrier's disease who suffered from severe hypoproteinemia and tested positive for Helicobacter pylori when initially admitted to hospital. Blood levels of intact glucagon-like peptide-2 (GLP-2) were determined by specific bioassay, using serum-induced cAMP production in COS-7 cells expressing recombinant human GLP-2 receptors as a functional readout. Eradication of H. pylori led to remission of Ménétrier's disease as well as a partial yet significant decrease in GLP-2 levels, and also improved hypoproteinemia. These observations suggest a possible link between excess systemic endogenous production of GLP-2, a gut hormone that induces mucosal growth, and the hypertrophic gastropathy in a Ménétrier's disease patient with H. pylori infection.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed2004

Interleukin-1 upregulates anaphylatoxin receptors on mononuclear cells.

BACKGROUND: The anaphylatoxins, C3a and C5a, that are generated during trauma, major surgery, or infection are potent proinflammatory mediators that increase interleukin (IL-1) cytokine synthesis. We investigated the effects of IL-1 on anaphylatoxin receptor expression in monocytes. METHODS: A human monocytic cell line, MONO-MAC-6, was used. C3a and C5a binding sites were assayed by competitive binding. Levels of messenger RNA for the C3a and C5a receptors were analyzed by reverse transcriptase-polymerase chain reaction. Changes of free cytosolic Ca(2+) concentration ([Ca(2+)]i) in response to C3a and C5a were measured. RESULTS: Basal MONO-MAC-6 cell sites for C3a and C5a binding were 10900 C3aR/cell (K(d)=2.0 nmol/L), 8700 C5aR/cell (K(d)=0.9 nmol/L). IL-1alpha increased sites for both C3a (61% increase; P <.01) and C5a (71% increase; P <.001). Levels of C3aR and C5aR messenger RNA also increased in IL-1alpha-stimulated cells. Receptors were coupled to functional responses, which were demonstrated by C3a- or C5a-induced [Ca(2+)]i increases. IL-1 receptor antagonist blocked the effects of IL-1alpha upregulation of anaphylatoxin receptors. CONCLUSION: These results suggest that there is an additional link between IL-1 and anaphylatoxins to amplify proinflammatory effects through monocytes and macrophages. Although C3a and C5a can increase the monocyte production of IL-1, IL-1 increases monocyte expression of receptors for these anaphylatoxins, which further amplifies inflammation.

Anaphylatoxins↗

PubMed2004

Ligand function at constitutively active receptor mutants is affected by two distinct yet interacting mechanisms.

It has been proposed that mutations that induce constitutive activity in G-protein-coupled receptors (GPCRs) concomitantly enhance the ability of partial agonists to trigger second-messenger signaling. Using the cholecystokinin type 2 receptor (CCK-2R) as a model system, we have explored whether this association applies to a diverse set of activating mutations. Consistent with established principles, constitutively active CCK-2Rs resulting from amino acid substitutions within the third intracellular loop each systematically increased partial agonist activities versus corresponding wild-type values. In contrast, activating mutations within transmembrane domain segments near the extracellular loops led to an increase in efficacy of only a subset of compounds but decreased or did not change the function of others. When transmembrane domain amino acid substitutions were introduced in combination with intracellular amplifying mutations, observed changes in ligand activity were defined by the product of two discernible factors 1) systematic amplification caused by an equilibrium shift from the inactive to the active receptor conformation and 2) ligand-specific alterations in signaling, which probably result from mutation-induced changes in the putative binding pocket. These findings illustrate functional heterogeneity among GPCR mutants with ligand-independent signaling. A subgroup of activating mutations facilitates receptor isomerization to the active state and in parallel perturbs ligand receptor interactions. These mutants do not adhere to the previously proposed "hallmark criteria" of constitutive activity.

Animals↗

PubMed2003

Established theory of radiation-induced decay is not generalizable to Bolton-Hunter labeled peptides.

Peptide hormones radiolabeled with 125I are widely used for the pharmacological characterization of cognate receptors. As a prerequisite for calculating ligand affinities from competition binding assays, and for estimating receptor densities from such studies, it is necessary to know the concentration of bioactive radioligand that is used in respective experiments. It has been demonstrated previously that radioiodinated peptides undergo decay catastrophe, i.e. disintegration of the radioactive label leads to the concomitant destruction of the carrier peptide. Here, we demonstrate that decay catastrophe does not apply to two peptide hormones that are iodinated by Bolton-Hunter conjugation: cholecystokinin octapeptide and glucagon-like peptide 2. The function of aged samples of these radioligands at corresponding recombinantly expressed receptors was assessed by measuring ligand-induced inositol phosphate production or generation of cyclic AMP, respectively. Both of the tested compounds, although predicted by decay catastrophe to contain little or subthreshold remaining bioactivity, stimulated an unexpectedly high level of receptor-mediated second messenger signaling. Quantitative comparison of observed functions with those of corresponding unlabeled peptides suggested that the bioactivity of each radioligand had been largely conserved despite the radioactive decay of the iodine label. Consistent with an apparent absence of decay catastrophe, we noted that the specific radioactivity, when determined immediately following peptide iodination, was close to the theoretical maximum but exponentially decreased over time. These findings raise the possibility that attachment of a Bolton-Hunter conjugate may shield labeled peptides from radiation-induced damage, a scenario that should be considered when performing radioligand binding experiments.

Animals↗

PubMed2003

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.

Animals↗

PubMed2003

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.

Alanine↗

PubMed2002

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.

Alternative Splicing↗