Lysine 173 Residue within the First Exoloop of Rat Secretin Receptor Is Involved in Carboxylate Moiety Recognition of Asp 3 in Secretin J. P. Vilardaga,* , ‡ E. Di Paolo,* P. De Neef,* M. Waelbroeck,* A.x Bollen,* , ‡ and P. Robberecht* ,1 *Department of Biochemistry and Nutrition, School of Medicine, Université Libre de Bruxelles, Bât. G/E, CP 611, 808 Route de Lennik, B 1070 Brussels, Belgium; and ²Department of Applied Genetics, ULB, 24 rue de l’Industrie, B 1400 Nivelles, Belgium Received November 30, 1995 The contribution of the extracellular loops of the secretin receptor to the recognition of secretin was inves- tigated by transfection in CHO cells of chimeric receptors, in which the three loops of the secretin recombinant receptor were replaced by the corresponding sequences of the glucagon receptor. The role of the third loop could not be evaluated as the transfected cells did not respond to secretin. Replacement of extracellular loop 2 reduced markedly the capability of secretin to occupy the receptor but did not alter the capacity of the receptor to discriminate between peptide analogues modified in position 3. Replacement of the first extracellular loop not only reduced the potency of secretin but also decreased the capacity of the receptor to discriminate between ligands having in position 3 an aspartate (as in secretin), an asparagine, or a glutamic acid. This change in receptor properties was reproduced by a single mutation of lysine 173 of the receptor into isoleucine. Thus, the basic amino acid in position 173 is likely to interact with aspartate 3 of secretin. As an aspartate is also present in position 3 of VIP and PACAP, two peptides related to secretin, and a lysine residue is conserved in the first extracellular loop of the VIP and PACAP receptors, this interaction may be a key element of peptide recognition by this receptor family. © 1996 Academic Press, Inc. The secretin receptor (Sn.r) [1], belongs to a subfamily within the G proteins coupled receptors family that includes also the mammalian receptors for VIP [2,3], PACAP [4], glucagon [5], glucagon-like-peptide I [6], growth hormone-releasing hormone [7], gastric inhibitory peptide [8], calcitonin [9], parathyroid hormone [10], corticotrophin-releasing factor [11] and an insect diuretic hormone [12]. There is a high overall sequence homology in the transmembrane domains. In contrast, the amino acid sequences of the N-terminal extracellular part of the receptors vary markedly, suggesting that this domain might contribute to the specific ligand recognition. It was recently demonstrated that the N-terminal extracellular part of the secretin receptor is indeed implicated in agonist discrimi- nation [13,14], but the precise contacts between the receptor and the ligand have not yet been identified. The aim of the present work was to study the possible role of the extracellular loops of the secretin receptor in ligand recognition. We took advantage of the absence of cross-recognition of secretin by the glucagon receptor or of glucagon by the secretin receptor (despite 40% identity and 60% similarity between both receptors), to construct glucagon/secretin chimeric receptors. We substituted each extracellular loop of the rat secretin receptor with the corresponding domain of the rat glucagon receptor, and generated stable CHO cell lines expressing the chimeric or wild-type proteins. Functional studies of chimeric receptors demonstrated that the first extracellular loop is impli- cated in the interaction with the Asp 3 residue of secretin. Further site-directed mutagenesis of that loop provided evidence that Lys 173 residue of the secretin receptor is responsible for that interac- tion. 1 Address for correspondence: Dr. P. Robberecht, Department of Biochemistry and Nutrition, School of Medicine, Université Libre de Bruxelles, Bât. G/E, CP 611, 808 Route de Lennik, B 1070 Brussels, Belgium. Fax: 32.2.555.62.30; Email: probbe@ulb.ac.be. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 218, 842–846 (1996) ARTICLE NO. 0150 842 0006-291X/96 $12.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.