Role of the N-terminal domain of the calcitonin receptor-like receptor in ligand binding

Madhu Chauhan, Krishna Rajarathnam, Chandra Yallampalli

Research output: Contribution to journalArticle

9 Citations (Scopus)

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-AM binding to pregnant rat uterine membrane in the presence of Nt-CRLR protein. We observe that Nt-CRLR inhibits 125I-CGRP and 125I-AM binding to rat uterus in a dose-dependent fashion (IC50 = 0.25 and 0.29 μM, 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.

Original languageEnglish (US)
Pages (from-to)782-789
Number of pages8
JournalBiochemistry
Volume44
Issue number2
DOIs
StatePublished - Jan 18 2005

Fingerprint

Calcitonin Receptor-Like Protein
Ligands
Adrenomedullin
Calcitonin Gene-Related Peptide
Proteins
Rats
Receptor Activity-Modifying Proteins
Adrenomedullin Receptors
Calcitonin Gene-Related Peptide Receptors
G-Protein-Coupled Receptors
Escherichia coli Proteins
Glucagon
Inclusion Bodies
Parathyroid Hormone
Escherichia coli
Inhibitory Concentration 50
Uterus
Fusion reactions
Fluorescence

ASJC Scopus subject areas

  • Biochemistry

Cite this

Role of the N-terminal domain of the calcitonin receptor-like receptor in ligand binding. / Chauhan, Madhu; Rajarathnam, Krishna; Yallampalli, Chandra.

In: Biochemistry, Vol. 44, No. 2, 18.01.2005, p. 782-789.

Research output: Contribution to journalArticle

Chauhan, Madhu ; Rajarathnam, Krishna ; Yallampalli, Chandra. / Role of the N-terminal domain of the calcitonin receptor-like receptor in ligand binding. In: Biochemistry. 2005 ; Vol. 44, No. 2. pp. 782-789.
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