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Patrick M Sexton

Publications and source records attributed to Patrick M Sexton.

31 records · Page 2Linked to original sources
PubMed2004

Regulation of serotonin 5-HT2C receptors by chronic ligand exposure.

The effect of ligand pretreatment on human 5-hydroxytryptamine2C (5-HT2C) receptors was examined in CHO cells expressing high (CHO-1C7; 67+/-3 pmol/mg) or low (CHO-1C19; 72+/-10 fmol/mg) levels of the receptor. Seventy-two hours pretreatment of CHO-1C7 cells with various ligands did not affect receptor expression. Pretreatment with inverse agonists enhanced 5-HT-mediated inositol phosphate accumulation with no change in constitutive receptor activity. The enhanced agonist responsiveness was inversely correlated with the intrinsic activity of the pretreatment ligand. Seventy-two hours of pretreatment with the weak agonist, 5-methoxygramine, caused an elevation in constitutive activity but no alteration in 5-HT-mediated signaling. In CHO-1C19 cells, 24 but not 72 h of pretreatment with the inverse agonist mianserin enhanced 5-HT-mediated signaling, with no effect on basal signaling; pretreatment with 5-methoxygramine had no significant effect. These findings highlight differences in the pattern of chronic regulation of 5HT2C receptor signaling between high and low receptor expression levels in a common cellular background.

Algorithms↗
PubMed2004

Regulation of M2 muscarinic acetylcholine receptor expression and signaling by prolonged exposure to allosteric modulators.

The effects of prolonged exposure of M(2) muscarinic acetylcholine receptors (mAChRs), stably expressed in Chinese hamster ovary cells, to the allosteric modulators gallamine, alcuronium, and heptane-1,7-bis (dimethyl-3'-phthalimidopropyl)-ammonium bromide (C(7)/3'-phth) were compared with the effects of the agonist carbachol (CCh) and antagonists atropine and N-methylscopolamine (NMS). Intact cell saturation binding assays using [(3)H]NMS found that pretreatment of the cells for 24 h with CCh caused a significant down-regulation of receptor number, whereas atropine, NMS, and all three allosteric modulators caused receptor up-regulation. Functional assays using a cytosensor microphysiometer to measure whole-cell metabolic rate found no acute effects of gallamine on receptor signaling, whereas atropine seemed to behave as an inverse agonist. Pretreatment of the cells with gallamine (20 microM) or atropine (20 nM) resulted in a significant enhancement of the maximal effect evoked by CCh. In contrast, CCh (100 microM) pretreatment resulted in a significant reduction in maximal receptor signaling capacity. Time-course experiments revealed that the effects of atropine and gallamine on receptor up-regulation are only visualized after at least 12-h ligand exposure, compared with the more rapid effects of CCh, which achieve steady-state down-regulation within 90 min. Additional experiments monitoring CCh-mediated M(2) mAChR internalization in the presence of gallamine revealed that part of the mechanism underlying the effects of the modulator on receptor expression may involve a change in receptor internalization properties. These findings suggest that, like orthosteric ligands, G protein-coupled receptor allosteric modulators also are able to mediate long-term effects on receptor regulation.

Alcuronium↗
PubMed2004

Application of a kinetic model to the apparently complex behavior of negative and positive allosteric modulators of muscarinic acetylcholine receptors.

The binding of allosteric modulators to G protein-coupled receptors (GPCRs) is often described by an equilibrium allosteric ternary complex model (ATCM). This study evaluated the effects of three modulators on the binding of [(3)H]N-methylscopolamine ([(3)H]NMS) to the human M(2) muscarinic acetylcholine receptor (mAChR). The binding of each modulator was more complex than predicted by the ATCM; the inhibitors heptane-1,7-bis-(dimethyl-3-phthalimidopropyl)-ammonium bromide and gallamine yielded biphasic curves that were described empirically by a two-site binding model, whereas the enhancer alcuronium yielded a bell-shaped curve. Radioligand dissociation assays revealed that the modulators retarded [(3)H]NMS kinetics such that the system never attained equilibrium. Subsequent application of a kinetic ATCM accommodated and quantified all experimental observations. Our findings confirm and extend previous studies on the use of a kinetic ATCM for mAChR allosteric enhancers, but also highlight how complex curves displayed by allosteric inhibitors can be misinterpreted in terms of multisite orthosteric binding. It is possible that similar behavior of other allosteric modulators at GPCRs may reflect nonequilibrium binding artifacts rather than deviation from an ATCM.

Allosteric Regulation↗
PubMed2004

Photoaffinity scanning in the mapping of the peptide receptor interface of class II G protein-coupled receptors.

The family of G protein-coupled receptors constitutes about 50% of the therapeutic drug targets used in clinical medicine today, although the mechanisms of ligand binding, activation and signal transduction for G protein-coupled receptors are not yet well defined. This review discusses ongoing research using the photoaffinity scanning method to map the bimolecular interface between class II G protein-coupled receptors and their ligands. Furthermore the available computer model of class II peptide ligand docking into the receptor, based on the positional constraints imposed by the photoaffinity scanning analyses, will be discussed briefly. The ultimate goal of these efforts is to understand the molecular basis of receptor binding and therefore to generate a template for rational drug design.

Amino Acid Sequence↗
PubMed2004

The receptor activity modifying protein family of G protein coupled receptor accessory proteins.

Receptor diversity for the calcitonin peptide family is created by the interaction of two 7-transmembrane proteins--the calcitonin receptor (CTR) or the calcitonin receptor-like receptor (CL-R)--with the receptor activity modifying protein (RAMP) family. The discovery of heterodimeric complexes of these proteins heralded a new era in the study of G protein coupled receptors (GPCRs), whereby receptor phenotype is no longer governed by just the GPCR. In this article, recent advances in the study of RAMPs are discussed--from our current understanding of the molecular basis of RAMP-receptor interaction to a broader role for RAMPs outside the calcitonin receptor family.

Amino Acid Sequence↗
PubMed2004

Calcitonin.

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Animals↗
PubMed2003

Spatial proximity between a photolabile residue in position 19 of salmon calcitonin and the amino terminus of the human calcitonin receptor.

Calcitonins are 32-amino acid peptide hormones with both peripheral and central actions mediated via specific cell surface receptors, which belong to the class II subfamily of G protein-coupled receptors. Understanding receptor function, particularly in terms of ligand recognition by calcitonin receptors, may aid in the rational design of calcitonin analogs with increased potency and improved selectivity. To directly identify sites of proximity between calcitonin and its receptor, we carried out photoaffinity labeling studies followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. A fully active salmon calcitonin analog [Arg(11,18),Bpa19]sCT, incorporating a photolabile p-benzoyl-L-phenylalanine into position 19 of the ligand, has been used to demonstrate spatial proximity between residue 19 of the peptide and the amino-terminal extracellular domain of the receptor. Cyanogen bromide cleavage together with endoproteinase Asp-N digestion indicated that binding was predominantly to the region delimited by receptor residues Cys134 and Met187. Binding to this fragment was supported further by cyanogen bromide-digestion of receptors that were mutated to remove the predicted cleavage site at Met133 (M133A, M133L). Binding within the 54-amino acid fragment was refined further by digestion with endoproteinase Lys-C to the 8-amino acid region corresponding to Cys134-Lys141. These results provide the first direct demonstration of a contact domain between salmon calcitonin and its receptor and will contribute toward modeling of the calcitonin-receptor interface.

Amino Acid Sequence↗
PubMed2003

RAMPs: 5 years on, where to now?

It is now approximately 5 years since the identification of the family of receptor activity modifying proteins (RAMPs). This finding revolutionized concepts of the pharmacology of G-protein-coupled receptors (GPCRs) and revealed that GPCR accessory proteins not only assist trafficking and folding but also define receptor type. Since the identification of RAMPs as modulators of the trafficking and properties of the calcitonin-receptor-like receptor, much work has focused on improving our understanding of the nature of RAMP-GPCR dimers, the extent to which they occur, and the consequence of this association. In this article, we review recent developments, including the identification of new receptor partners and novel roles for RAMPs.

Calcitonin Receptor-Like Protein↗
PubMed2002

Novel receptor partners and function of receptor activity-modifying proteins.

The receptor activity-modifying proteins (RAMPs) comprise a family of three accessory proteins that heterodimerize with the calcitonin receptor-like receptor (CL receptor) or with the calcitonin receptor (CTR) to generate different receptor phenotypes. However, RAMPs are more widely distributed across cell and tissue types than the CTR and CL receptor, suggesting additional roles for RAMPs in cellular processes. We have investigated the potential for RAMP interaction with a number of Class II G protein-coupled receptors (GPCRs) in addition to the CL receptor and the CTR. Using immunofluorescence confocal microscopy, we demonstrate, for the first time, that RAMPs interact with at least four additional receptors, the VPAC1 vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor with all three RAMPs; the glucagon and PTH1 parathyroid hormone receptors with RAMP2; and the PTH2 receptor with RAMP3. Unlike the interaction of RAMPs with the CL receptor or the CTR, VPAC1R-RAMP complexes do not show altered phenotypic behavior compared with the VPAC1R alone, as determined using radioligand binding in COS-7 cells. However, the VPAC1R-RAMP2 heterodimer displays a significant enhancement of agonist-mediated phosphoinositide hydrolysis with no change in cAMP stimulation compared with the VPAC1R alone. Our findings identify a new functional consequence of RAMP-receptor interaction, suggesting that RAMPs play a more general role in modulating cell signaling through other GPCRs than is currently appreciated.

Animals↗
PubMed2002

Lipopolysaccharide supports survival and fusion of preosteoclasts independent of TNF-alpha, IL-1, and RANKL.

Lipopolysaccharide (LPS), a cell component of Gram-negative bacteria, is a pathogen of inflammatory bone loss. To examine the effects of LPS on the survival and fusion of osteoclasts, mononuclear osteoclasts (preosteoclasts, pOCs) were collected from a mouse co-culture system and cultured in the presence or absence of LPS. Most pOCs died within 24 h in the absence of any stimulus. LPS as well as receptor activator of NF-kappaB ligand (RANKL) supported the survival of pOCs, and induced their fusion to form multinucleated cells (MNCs). Like authentic osteoclasts, MNCs induced by LPS expressed calcitonin receptors, and formed actin rings on culture plates. LPS-induced MNC formation in pOC cultures was observed even in the presence of osteoprotegerin and interleukin (IL)-1-receptor antagonists. MNC formation was also stimulated by LPS in pOC cultures prepared from tumor necrosis factor (TNF)-receptor-I or TNF-receptor-II deficient mice. LPS induced the degradation of IkappaB in pOCs within 20 min. Lactacystin, an inhibitor of NF-kappaB activation, and wortmannin, an inhibitor of phosphatidylinositol-3 kinase, strongly inhibited LPS-induced MNC formation in pOC cultures. LPS induced pit-forming activity of pOCs in the presence of macrophage-colony stimulating factor (M-CSF). These findings suggest that LPS stimulates the survival and fusion of pOCs, independent of RANKL, IL-1 or TNF-alpha action. Activation of NF-kappaB and phosphatidylinositol-3 kinase appeared to be involved in LPS-induced effects on pOCs. These observations suggest that LPS is involved directly in inflammatory bone loss, and also indirectly through the production of LPS-induced host factors such as IL-1 and TNF-alpha.

Animals↗
PubMed2002

International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors.

The calcitonin family of peptides comprises calcitonin, amylin, two calcitonin gene-related peptides (CGRPs), and adrenomedullin. The first calcitonin receptor was cloned in 1991. Its pharmacology is complicated by the existence of several splice variants. The receptors for the other members the family are made up of subunits. The calcitonin-like receptor (CL receptor) requires a single transmembrane domain protein, termed receptor activity modifying protein, RAMP1, to function as a CGRP receptor. RAMP2 and -3 enable the same CL receptor to behave as an adrenomedullin receptor. Although the calcitonin receptor does not require RAMP to bind and respond to calcitonin, it can associate with the RAMPs, resulting in a series of receptors that typically have high affinity for amylin and varied affinity for CGRP. This review aims to reconcile what is observed when the receptors are reconstituted in vitro with the properties they show in native cells and tissues. Experimental conditions must be rigorously controlled because different degrees of protein expression may markedly modify pharmacology in such a complex situation. Recommendations, which follow International Union of Pharmacology guidelines, are made for the nomenclature of these multimeric receptors.

Animals↗
PubMedDate not supplied

"Ins and outs" of seven-transmembrane receptor signalling to ERK.

Extracellular-signal-regulated kinases 1 and 2 (ERK1/2) are important members of the mitogen-activated protein kinase (MAPK) family and have emerged as key effector targets of activation by seven-transmembrane-spanning (G-protein-coupled) receptors (7TMRs). Regulation of ERK by 7TMRs is highly complex and dependent on cell type. Numerous studies have linked specific G protein pathways to ERK activation, but recent evidence suggests that some 7TMR-linked ERK signalling pathways might not be exclusively mediated by G proteins. In addition, the emergence of an "inside-out" model for receptor tyrosine kinase (RTK) "transactivation" by 7TMRs has enhanced our understanding of the ERK signalling system and further underscores the complexity of mitogenic regulation by 7TMRs.

Animals↗