A SINGLE AMINO-ACID SUBSTITUTION IN SOMATOSTATIN RECEPTOR SUBTYPE-5 INCREASES AFFINITY FOR SOMATOSTATIN-14

Four of the five somatostatin receptor (SSTR) subtypes bind the two na tive forms of somatostatin, i.e., somatostatin-14 (S-14) and amino-ter minally extended somatostatin-28 (S-28), with comparable affinities (a pproximately 0.2 nM). The SSTR5 subtype exhibits 10-50-fold higher aff inity for S-28 than for S-14 (0.2 and 5 nM, respectively). To determin e which domains in SSTR5 are responsible for the observed pharmacologi cal 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 cri tical role in the lower binding affinity of S-14 for SSTR5. Substituti on of this region with the corresponding region of SSTR2 produced chim eric 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 pos itions in SSTR5, these residues are glycine (G258) and phenylalanine ( F265), respectively. Substitution of G258 with phenylalanine did not a lter 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-fo ld, 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 aboli sh the preference for S-28 over S-14. This finding suggests that the t yrosine in the predicted TM6 may be an important contact point between somatostatin and SSTR.