Application of photoaffinity crosslinking in determining the interaction between calcitonin and its receptor Vi Pham, John D. Wade & Patrick M. Sexton* Howard Florey Institute, The University of Melbourne, Gate 11, Royal Parade, Parkville, Victoria 3010, Australia (*Author for correspondence, e-mail: p.sexton@hfi.unimelb.edu.au, Tel: 61 3 8344 7334, Fax: 61 3 9348 1707) Received 22 December 2003; Accepted 02 March 2004 Key words: calcitonin, calcitonin receptor, class II G protein-coupled receptor, ligand-receptor interac- tion, photoaffinity cross-linking Summary Understanding the molecular mechanism underlying how the peptide ligands bind to their receptors with subsequent receptor activation and cellular response is of great long-term value in designing receptor- targeted drugs. This is more difficult for class-II G protein-coupled receptors as only minimal structural data is available and their natural peptide ligands contain a large and diffuse pharmacophore. To address this problem, photoaffinity labeling studies have been developed to identify the spatial proximity between the photophore-modified ligand and its receptor. This minireview looks at the application of this approach in determining the proximal sites between class-II G protein-coupled receptor peptide ligands and their corresponding receptors, including parathyroid hormone, secretin and vasoactive intes- tinal polypeptide. More specifically, we will highlight interaction sites between positions 19, 16 and 26 of calcitonin with C 134 –K 141 , and F 137 and T 30 of the receptor, respectively. Abbreviations: Bpa, p-benzoyl-L-phenylalanine; CNBr, cyanogen bromide; CT, calcitonin; CTR, calcito- nin receptor; DMSO, dimethyl sulfoxide; ECD, extracellular domain; ECL, extracellular loop; Endo-F, endoglycosydase F; GPCRs, guanine nucleotide-binding protein (G protein)-coupled receptors; h, human; ICL, intracellular loop; Lys-C, endoproteinase Lys-C; MALDITOF, matrix-assisted laser desorption time of flight; NMR, nuclear magnetic resonance; pBz 2 , e-p-benzoylbenzoyl; PTH, parathy- roid hormone; PTHrP, parathyroid hormone-related protein; RP-HPLC, reverse-phase high perfor- mance liquid chromatography; s, salmon; TFE, trifluoroethanol; TMH, transmembrane-spanning helix; VIP, vasoactive intestinal peptide. Introduction Guanine nucleotide-binding protein (G protein)- coupled receptors (GPCRs) are the largest family of plasma membrane receptor proteins. They are characterised by a N-terminal extracellular domain (ECD), seven hydrophobic transmem- brane-spanning helices (TMHs) connected by three hydrophilic extracellular loops (ECLs) and three hydrophilic intracellular loops (ICLs), and an intracellular carboxyl terminus [1]. Based on overall structural homology, GPCRs can be sub- divided into three major receptor families [2]. The second largest GPCR class (class-II or fam- ily B) includes receptors for peptide hormones such as calcitonin, secretin and parathyroid hor- mone (PTH). These class-II peptide hormone receptors share 30–50% sequence identity and have a number of conserved structural fea- tures including (1) a large N-terminal ECD (150– 180 amino acids) that includes six conserved cysteine residues and several potential N-linked Letters in Peptide Science, 10: 447–453, 2003. KLUWER / ESCOM 447 Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands.