ENDOCYTOSIS OF THE RAT SOMATOSTATIN RECEPTORS - SUBTYPE DISCRIMINATION, LIGAND SPECIFICITY, AND DELINEATION OF CARBOXY-TERMINAL POSITIVE AND NEGATIVE SEQUENCE MOTIFS

Endocytosis of the five rat somatostatin receptor subtypes (SSTR1-5) w as investigated in transfected HEK cells by biochemical ligand binding assays and confocal microscopic analysis. Phenylarsine oxide-sensitiv e internalization of SSTR1-3 is dependent on SST-14 or SST-28, whereas only the octacosapeptide triggers this reaction with SSTR5. SSTR4 is not internalized with either SST, Internalized SSTR3 is cycled back to the plasma membrane while endocytosed rho-Ala(1)-SST-14 remains insid e the cell, Delineation of sequence motifs responsible for internaliza tion of SSTR3 revealed multiple serines and a threonine (Ser-341, Ser- 346, Ser-351, and Thr-357) within the carboxy-terminal tail of which S er-351 and Thr-357 were the most effective ones, Chimeras in which var ious segments of the carboxyl terminus of SSTR4 were replaced by the c orresponding regions of SSTR3 were internalized as long as they contai n the Ser/Thr motif, However, this internalization reaction was suppre ssed when the chimeras were extended by the carboxyl terminus of SSTR4 (residues 320-384), suggesting the presence of a negative control ele ment in that region, Step-wise truncation of the carboxyl terminus of wild-type SSTR4 revealed a motif of three amino acid residues Glu-Thr- Thr (SSTR4-330-332) that is responsible for preventing internalization and may be important in regulating endocytosis of this receptor subty pe.