LIGAND-BINDING POCKET OF THE HUMAN SOMATOSTATIN RECEPTOR-5 - MUTATIONAL ANALYSIS OF THE EXTRACELLULAR DOMAINS

The ligand binding domain of G protein-coupled receptors for peptide l igands consists of a pocket formed by extracellular and transmembrane domain (TM) residues. In the case of somatostatin (SRIF), however, pre vious studies have suggested that the binding cavity of the octapeptid e analog SMS201-995 (SMS) is lined by residues in TMs III-VII. The add itional involvement of the extracellular domains for binding SMS or th e natural SRIF ligands (SRIF-14, SRIF-28) has not been clarified. Usin g a cassette construct cDNA for the human somatostatin 5 receptor (sst (5)R), we systematically examined the role of exofacial structures in ligand binding by creating a series of mutants in which the extracellu lar portions have been altered by conservative segment exchange (CSE) mutagenesis for the extracellular loops (ECLs) and by deletion (for th e NH2-terminal segment) or truncation analysis (ECL3). CHO-K1 cells we re stably transfected with wild type or mutant human sst(5)R construct s, and agonist binding was assessed using membrane binding assays with I-125-LTT SRIF-28 ligand. Deletion of the NH2 terminus or CSE mutagen esis of ECL1 and ECL3 produced minor 2-8-fold decreases in affinity fo r SRIF-14, SRIF-28, and SMS ligands. Truncation of ECL3 to mimic the s ize of this loop in sst(1)R and sst(4)R (the two subtypes that do not bind SMS) did not interfere with the binding of SMS, SRIF-14, or SRIF- 28. In contrast, both ECL2 mutants failed to bind I-125-LTT SRIF-28. I mmunocytochemical analysis of nonpermeabilized cells with a human sst( 5)R antibody revealed that the mutant receptors were targeted to the p lasma membrane. Labeled SMS (I-125-Tyr3 SMS) also failed to bind to th e mutant ECL2 receptors. These results suggest a potential contributio n of ECL2 (in addition to the previously identified residues in TMs II I-VII) to the SRIF ligand binding pocket.