2 AMINO-ACIDS, LOCATED IN TRANSMEMBRANE DOMAIN-VI AND DOMAIN-VII, DETERMINE THE SELECTIVITY OF THE PEPTIDE AGONIST SMS-201-995 FOR THE SSTR2 SOMATOSTATIN RECEPTOR

Human somatostatin receptor subtypes (SSTR1-5) bind their natural liga nds SRIF-14 and SRIF-28 with high affinity, By contrast, short synthet ic SRIF analogues such as SMS 201-995, a peptide agonist used for the treatment of various endocrine and malignant disorders, display sub-na nomolar affinity only for the receptor subtype SSTR2. To understand th e molecular nature of selective peptide agonist binding to somatostati n receptors we have now, by site-directed mutagenesis, identified amin o acids mediating SMS 201-995 specificity for SSTR2. Sequentially, ami no acids in SSTR1, a receptor subtype exhibiting low affinity for SMS 201-995, were exchanged for the corresponding SSTR2 residues, After th ree consecutive steps, in which eight amino acids were exchanged, a SS TR1 mutant receptor with high affinity for SMS 201-995 was obtained. R eceptor mutants with different combinations of these eight amino acids were then constructed, A single Ser305 to Phe mutation in TM VII incr eased the affinity of SSTR1 for SMS 201-995 nearly 100-fold, When this mutation was combined with an exchange of Gln291 to Asn in TM VI, alm ost full susceptibility to SMS 201-995 was obtained, Thus, it is concl uded that the specificity of SMS 201-995 for SSTR2 is mainly defined b y these two amino acids in transmembrane domains VI and VII. Using the conjugate gradient method we have, by analogy to the well established structure of bacteriorhodopsin, built a model for SRIF receptor-ligan d interactions that explains the importance of Gln291 and Ser305 for t he selectivity of agonists.