THE CYTOPLASMIC TAIL OF THE HUMAN SOMATOSTATIN RECEPTOR-TYPE-5 IS CRUCIAL FOR INTERACTION WITH ADENYLYL-CYCLASE AND IN MEDIATING DESENSITIZATION AND INTERNALIZATION

We have investigated the role of the cytoplasmic tail (C-tail) of the human somatostatin receptor type 5 (hSSTR5) in regulating receptor cou pling to adenylyl cyclase (AC) and in mediating agonist-dependent dese nsitization and internalization responses. Mutant receptors with progr essive C-tail truncation (Delta 347, Delta 338, Delta 328, Delta 318), Cys(320) --> Ala substitution (to block palmitoylation), or Tyr(304) --> Ala substitution of a putative NPXYY internalization motif were st ably expressed in Chinese hamster ovary K1 cells. Except for the Tyr(3 04) --> Ala mutant, which showed no binding, all other mutant receptor s exhibited binding characteristics (K-d and B-max) and G protein coup ling comparable with wild type (wt) hSSTR5. The C-tail truncation muta nts displayed progressive reduction in coupling to AC, with the Delta 318 mutant showing complete loss of effector coupling. Agonist pretrea tment of wt hSSTR5 led to uncoupling of AC inhibition, whereas the des ensitization response of the C-tail deletion mutants was variably impa ired. Compared with internalization (66% at 60 min) of wt hSSTR5, trun cation of the C-tail to 318, 328, and 338 residues reduced receptor in ternalization to 46, 46, and 23%, respectively, whereas truncation to 347 residues slightly improved internalization (72%). Mutation of Cys( 320) --> Ala induced a reduction in AC coupling, desensitization, and internalization. These studies show that the C-tail of hSSTR5 serves a multifunctional role in mediating effector coupling, desensitization, and internalization. Whereas coupling to AC is dependent on the lengt h of the C-tail, desensitization and internalization require specific structural domains. Furthermore, internalization is regulated through both positive and negative molecular signals in the C-tail and can be dissociated from the signaling and acute desensitization responses of the receptor.