ABSTRACT
The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of two membrane proteins: calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). CLR is a class B G-protein-coupled receptor (GPCR), possessing a characteristic large amino-terminal extracellular domain (ECD) important for ligand recognition and binding. Dimerization of CLR with RAMP1 provides specificity for CGRP versus related agonists. Here we report the expression, purification, and refolding of a soluble form of the CGRP receptor comprising a heterodimer of the CLR and RAMP1 ECDs. The extracellular protein domains corresponding to residues 23-133 of CLR and residues 26-117 of RAMP1 were shown to be sufficient for formation of a stable, monodisperse complex. The binding affinity of the purified ECD complex for the CGRP peptide was significantly lower than that of the native receptor (IC(50) of 12 microM for the purified ECD complex vs 233 pM for membrane-bound CGRP receptor), indicating that other regions of CLR and/or RAMP1 are important for peptide agonist binding. However, high-affinity binding to known potent and specific nonpeptide antagonists of the CGRP receptor, including olcegepant and telcagepant (K(D) < 0.02 muM), as well as N-terminally truncated peptides and peptide analogues (140 nM to 1.62 microM) was observed.
Subject(s)
Extracellular Space/chemistry , Protein Folding , Receptors, Calcitonin Gene-Related Peptide/chemistry , Receptors, Calcitonin/chemistry , Amino Acid Sequence , Binding, Competitive , Calcitonin Receptor-Like Protein , Cell Line, Tumor , Crystallography, X-Ray , Dimerization , Extracellular Space/metabolism , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Ligands , Macromolecular Substances/chemistry , Macromolecular Substances/metabolism , Magnetic Resonance Spectroscopy , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Receptor Activity-Modifying Protein 1 , Receptor Activity-Modifying Proteins , Receptors, Calcitonin/metabolism , Receptors, Calcitonin Gene-Related Peptide/biosynthesis , Receptors, Calcitonin Gene-Related Peptide/genetics , Receptors, Calcitonin Gene-Related Peptide/isolation & purification , SolubilityABSTRACT
Functional binding sites for [125I]IAPP and [125I]CGRP were solubilized from rat lung membranes with CHAPSO (10 mM). Rat IAPP had a higher affinity (Ki = 22.9 nM) for [125I]IAPP binding and rat alpha CGRP (Ki = 0.904 nM) had a higher affinity for [125I]CGRP binding over related peptides. [125I]IAPP binding was unaffected by GTP gamma S, but [125I]CGRP binding was 50% inhibited, indicating solubilization of a G-protein-receptor complex for CGRP but not IAPP binding. Wheat germ agglutinin affinity columns gave a 25-fold purification of IAPP binding sites, but no CGRP binding sites were eluted from the column, indicating different patterns of glycosylation of the two sites.
Subject(s)
Amyloid/metabolism , Calcitonin Gene-Related Peptide/metabolism , Lung/metabolism , Receptors, Calcitonin Gene-Related Peptide/metabolism , Receptors, Peptide/metabolism , Animals , Binding Sites , Binding, Competitive , Cell Membrane/metabolism , Cholic Acids , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Islet Amyloid Polypeptide , Kinetics , Molecular Weight , Rats , Receptors, Calcitonin Gene-Related Peptide/isolation & purification , Receptors, Islet Amyloid Polypeptide , Receptors, Peptide/isolation & purification , SolubilityABSTRACT
The autoradiographic localization of binding sites for [125I]BH-[Sar9,Met(O2)11]SP, [125I]NKA, and [125I]CGRP was investigated in adjacent sections of urinary bladder body, from adult rats pretreated 14 days before with capsaicin or vehicle. Location of silver grains was assessed both qualitatively and quantitatively using computerized densitometry. Dense labeling of smooth muscle was seen with both [125I]BH-[Sar9,Met(O2)11]SP ([125I]BHSar-SP) and [125I]NKA; in addition, [125I]BHSar-SP labeled submucosal blood vessels. For these radioligands, no differences were apparent between sections from capsaicin- and vehicle-pretreated rats. Specific binding of [125I]CGRP was observed over the epithelium and weakly over submucosal arterioles, but not over smooth muscle. The density of [125I]CGRP binding sites on the epithelium, but not blood vessels, was increased (p < 0.05) by 22% after chronic capsaicin pretreatment, suggesting receptor upregulation. This study demonstrates that although all three peptides are colocalized in primary afferent sensory fibers in rat urinary bladder, the receptors for these neuropeptides are located on different cell types and may be subject to different neural influences.
Subject(s)
Capsaicin/pharmacology , Receptors, Calcitonin Gene-Related Peptide/isolation & purification , Receptors, Tachykinin/isolation & purification , Urinary Bladder/chemistry , Animals , Autoradiography , Calcitonin Gene-Related Peptide/metabolism , Male , Neurokinin A/metabolism , Protein Binding , Rats , Rats, Wistar , Receptors, Calcitonin Gene-Related Peptide/drug effects , Receptors, Tachykinin/drug effects , Substance P/analogs & derivatives , Substance P/metabolism , Tissue Distribution , Urinary Bladder/drug effectsABSTRACT
Intact calcitonin gene-related peptide (CGRP) receptors were solubilized from porcine neural membranes using sodium cholate: potassium buffer. The solubilized receptors were purified sequentially by hydrophobic interaction and ion-exchange chromatography followed by specific affinity chromatography. Using these procedures, we have isolated 2 nmol of highly purified active CGRP receptor to a homogeneity (5 x 10(8)-fold purification). The isolated receptors retained their specificity and the capacity to bind to 125I-CGRP, and showed no cross-reactivity with a number of other peptides, except with amylin having 46% amino acid sequence homology to h-CGRP. The solubilized receptors were adsorbed by WGA-agarose and concanavalin-A, suggesting a glycoprotein nature. SDS-PAGE, size-exclusion HPLC, and autoradiography confirmed that CGRP receptor is a monomeric membrane protein with M(r) 66 kDa.
