Abstract

Four of the five somatostatin receptor (SSTR) subtypes bind the two native forms of somatostatin, i.e., somatostatin-14 (S-14) and amino-terminally extended somatostatin-28 (S-28), with comparable affinities (approximately 0.2 nM). The SSTR5 subtype exhibits 10-50-fold higher affinity for S-28 than for S-14 (0.2 and 5 nM, respectively). To determine which domains in SSTR5 are responsible for the observed pharmacological selectivity, a series of SSTR2/SSTR5 chimeras were constructed and expressed in Chinese hamster ovary cells. Saturation and competition radioligand binding studies demonstrated that the region encompassing transmembrane domain 6 (TM6) through the carboxyl terminus plays a critical role in the lower binding affinity of S-14 for SSTR5. Substitution of this region with the corresponding region of SSTR2 produced chimeric receptors with high affinity for both S-28 and S-14. Examination of amino acid sequences revealed both a specific conserved hydrophobic residue and a conserved tyrosine in TM6 of SSTR1-4. At comparable positions in SSTR5, these residues are glycine (G258) and phenylalanine (F265), respectively. Substitution of G258 with phenylalanine did not alter the preference of SSTR5 for S-28 over S-14. However, substitution of F265 with tyrosine increased the binding affinity of S-14 by 20-fold, to an affinity comparable to that observed for the SSTR2 subtype. These data indicate that replacement of phenylalanine with tyrosine at position 265 in SSTR5 can modify ligand binding selectivity and abolish the preference for S-28 over S-14. This finding suggests that the tyrosine in the predicted TM6 may be an important contact point between somatostatin and SSTR.