《Role of the N-terminal domain of the calcitonin receptor-like receptor in ligand binding》.pdf

782 Biochemistry 2005, 44, 782789 Role of the N-Terminal Domain of the Calcitonin Receptor-like Receptor in Ligand Binding† Madhu Chauhan,‡ Krishna Rajarathnam,*,§ and Chandra Yallampalli*,‡ Departments of Obstetrics and Gynecology and Human Biological Chemistry and Genetics, Sealy Center for Structural Biology, Uniersity of Texas Medical Branch at Galeston, Galeston, Texas 77555 Recei ed April 27, 2004; Reised Manuscript Receied August 26, 2004 ABSTRACT : 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 125I-CGRP and 125I-