ASPARTIC-ACID RESIDUE-124 IN THE THIRD TRANSMEMBRANE DOMAIN OF THE SOMATOSTATIN RECEPTOR SUBTYPE-3 IS ESSENTIAL FOR SOMATOSTATIN-14 BINDING

A highly conserved aspartic acid residue present in the third membrane -spanning region of adrenergic and muscarinic receptors is directly in volved in ligand binding. The five cloned somatostatin receptor subtyp es also contain this residue at the same relative position. To test wh ether Asp-124 of the rat somatostatin receptor subtype 3 (SSTR3) is re sponsible for the binding of somatostain-14 (SST-14), this amino acid residue was replaced by an asparagine or a glutamic acid by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Expression a nd binding activity of the wild-type and mutant receptor constructs we re studied in COS and HEK cells by ligand binding, UV cross-linking, W estern blot, and immunocytochemical analysis. The Asn or Glu mutations result in a significant loss of SST-14 binding, although the mutant r eceptors are correctly transferred to the cell surface, demonstrating that Asp-124 is directly involved in binding of SST-14.