ROLE OF THE C-TERMINUS IN HISTAMINE-H2-RECEPTOR SIGNALING, DESENSITIZATION, AND AGONIST-INDUCED INTERNALIZATION

To evaluate the role of the histamine H2 receptor C terminus in signal ing, desensitization, and agonist-induced internalization, canine H2 r eceptors with truncated C termini were generated. Wild-type (WT) and t runcated receptors were tagged at their N termini with a hemagglutinin (HA) epitope and expressed in COS7 cells, Most of the C-terminal intr acellular tail could be truncated (51 of 70 residues, termed T-308 mut ant) without loss of functions: cAMP production, tiotidine binding, an d plasma membrane targeting, In fact, the T-308 mutant produced more c AMP than the WT when cell-surface expression per cell was equivalent. Pretreat ment of cells with 10(-5) M histamine desensitized cAMP produ ctions via WT and T-308 receptors 60 similar extents. Incubation of ce lls expressing WT receptors with 10(-5) M histamine reduced cell-surfa ce anti-HA antibody binding by approximately 30% (by 30 min, t(1/2) si milar to 15 min), but did not affect the B-max of tiotidine in membran e fractions, which represents total receptor amounts, suggesting that WT receptors were internalized from the cell surface, In contrast, no internalization was observed with T-308 receptors following histamine treatment. A mutant with a deletion of the 30 C-terminal amino acids, termed T-329, was functional but was as potent as the WT in terms of c AMP production. Apart from being desensitized by histamine, the intern alization of the receptor was indistinguishable from that of the WT. I nternalization was observed in the T-320 but not in T-313 mutant, narr owing the region involved in internalization to that between Glu(314) and Asn(320) (ETSLRSN). Of these seven residues, either Thr(315), Ser( 316), or both, were replaced with Ala. Thr(315) and Ser(316) are conse rved among species. The mutation at Thr(315) (but not that at Ser(316) ) abolished internalization. Taken together, these results demonstrate that Thr(315) is involved in agonist-induced internalization. Further more, the finding that T-308 receptors were desensitized in the absenc e of internalization suggests that internalization and desensitization are meditated by independent mechanisms.