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 ligands SRIF-14 and SRIF-28 with high affinity, By contrast, short synthetic SRIF analogues such as SMS 201-995, a peptide agonist used for the treatment of various endocrine and malignant disorders, display sub-nanomolar affinity only for the receptor subtype SSTR2. To understand the molecular nature of selective peptide agonist binding to somatostatin receptors we have now, by site-directed mutagenesis, identified amino acids mediating SMS 201-995 specificity for SSTR2. Sequentially, amino acids in SSTR1, a receptor subtype exhibiting low affinity for SMS 201-995, were exchanged for the corresponding SSTR2 residues, After three consecutive steps, in which eight amino acids were exchanged, a SSTR1 mutant receptor with high affinity for SMS 201-995 was obtained. Receptor mutants with different combinations of these eight amino acids were then constructed, A single Ser305 to Phe mutation in TM VII increased 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, almost full susceptibility to SMS 201-995 was obtained, Thus, it is concluded that the specificity of SMS 201-995 for SSTR2 is mainly defined by 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-ligand interactions that explains the importance of Gln291 and Ser305 for the selectivity of agonists.