Role of the N-terminal domain of the calcitonin receptor-like receptor in ligand binding.
Calcitonin receptor-like receptor (CRLR) is a seven-transmembrane (7-TM) domain class B G protein-coupled receptor (GPCR) which requires coexpression of different receptor activity modifying proteins (RAMP) to become a functional calcitonin gene-related peptide (CGRP) receptor or an adrenomedullin (AM) receptor. The N-terminal (Nt) extracellular region of class B GPCRs in ligand binding has been reported for receptors such as glucagon and parathyroid hormone. We hypothesize that the Nt-domain of CRLR (Nt-CRLR) is an autonomously folded unit possessing a well-defined structure and is involved in ligand binding and specificity. To obtain structural and functional information on the Nt-CRLR, we cloned and expressed the Nt-CRLR as a fusion protein in Escherichia coli. Overexpressed protein formed an inclusion body, which was refolded and purified, resulting in a soluble monomeric protein. Far-UV CD and fluorescence spectra of Nt-CRLR showed characteristics of a folded protein. The ability of Nt-CRLR to bind CGRP and AM independent of RAMPs was determined by studying inhibition of (125)I-CGRP and (125)I-AM binding to pregnant rat uterine membrane in the presence of Nt-CRLR protein. We observe that Nt-CRLR inhibits (125)I-CGRP and (125)I-AM binding to rat uterus in a dose-dependent fashion (IC(50) = 0.25 and 0.29 muM, respectively). Taken together, our data provide evidence that Nt-CRLR is structured and further that a significant part of the binding affinity comes from binding to the Nt-domain.
Chauhan
Madhu
M
Departments of Obstetrics and Gynecology, University of Texas Medical Branch at Galveston, Galveston, Texas 77555, USA.
Rajarathnam
Krishna
K
Yallampalli
Chandra
C
D053607:Adrenomedullin; D000818:Animals; D001667:Binding, Competitive; D015740:Calcitonin Gene-Related Peptide / Q000037:antagonists & inhibitors / Q000378:metabolism; D058286:Calcitonin Receptor-Like Protein; D002942:Circular Dichroism; D003001:Cloning, Molecular; D005260:Female; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010455:Peptides / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D018003:Receptors, Calcitonin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014461:Ultracentrifugation; D014599:Uterus / Q000737:chemistry / Q000378:metabolism
C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; C545580:Calcrl protein, rat; D007457:Iodine Radioisotopes; D008024:Ligands; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D018003:Receptors, Calcitonin; D053607:Adrenomedullin; D015740:Calcitonin Gene-Related Peptide