IDENTIFICATION OF LIGAND-BINDING DETERMINANTS IN THE SOMATOSTATIN RECEPTOR SUBTYPE-1 AND SUBTYPE-2

The somatostatin (SRIF) receptors (SSTRs) 1 and 2 bind SRIF and SRIF 2 8 with high affinity, although a number of synthetic hexapeptide and o ctapeptide analogs of SRIF bind selectively to SSTR2. Extracellular lo op three and its adjoining trans-membrane-spanning regions contain ele ments essential for the binding of such analogs to murine SSTR2. In pa rticular, a stretch of amino acids from residues 294-297 (FDFV) in mur ine SSTR2 in trans membrane domain seven can determine affinity for th e SSTR2-selective analogs. Within this region, Phe(294) has previously been predicted to be essential for the binding of octapeptides (Kaupm ann, K., Bruns, C., Raulf, F., Weber, H., Mattes, H., and Lubbert, H. (1995) EMBO J. 14, 727-735) based on the observation that SSTR1 can bi nd the octapeptide SMS-201-995 with reasonable affinity after a Ser to -Phe conversion in the analogous region of this receptor (SSTR(1S305F) ). We find that SSTR1(S305F) has low affinity for a number of SSTR2-se lective hexapeptides, suggesting that these analogs have different bin ding requirements than SMS-201-995. A correlation is seen between the ability of SSTR1(S305F) to bind hexapeptide analogs and the presence o f a phenylalanine, but not tyrosine, at position two in these small cy clic molecules. Thus, a single hydroxyl group in hexapeptides can play a critical role in determining re ceptor binding to these receptor mu tants. We also find that the second extracellular loop of SSTR1 is imp ortant for the selectivity of certain SRIF agonists for binding to SST R1. Taken together, our data indicate that there are multiple elements in the somatostatin receptors that can determine the binding affinity and selectivity of peptide analogs.