Refolding and characterization of a soluble ectodomain complex of the calcitonin gene-related peptide receptor.
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.
Koth
Christopher M
CM
Vertex Pharmaceuticals Inc., 130 Waverly Street, Cambridge, Massachusetts 02139, USA.
Abdul-Manan
Norzehan
N
Lepre
Christopher A
CA
Connolly
Peter J
PJ
Yoo
Sanghee
S
Mohanty
Arun K
AK
Lippke
Judith A
JA
Zwahlen
Jacque
J
Coll
Joyce T
JT
Doran
John D
JD
Garcia-Guzman
Miguel
M
Moore
Jonathan M
JM
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D058286:Calcitonin Receptor-Like Protein; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D019281:Dimerization; D005110:Extracellular Space / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000737:chemistry* / Q000378:metabolism; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins; C545502:RAMP1 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D018015:Receptors, Calcitonin Gene-Related Peptide