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 signaling, desensitization, and agonist-induced internalization, canine H2 receptors with truncated C termini were generated. Wild-type (WT) and truncated receptors were tagged at their N termini with a hemagglutinin (HA) epitope and expressed in COS7 cells, Most of the C-terminal intracellular tail could be truncated (51 of 70 residues, termed T-308 mutant) without loss of functions: cAMP production, tiotidine binding, and plasma membrane targeting, In fact, the T-308 mutant produced more cAMP than the WT when cell-surface expression per cell was equivalent. Pretreat ment of cells with 10(-5) M histamine desensitized cAMP productions via WT and T-308 receptors 60 similar extents. Incubation of cells expressing WT receptors with 10(-5) M histamine reduced cell-surface anti-HA antibody binding by approximately 30% (by 30 min, t(1/2) similar to 15 min), but did not affect the B-max of tiotidine in membrane 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 cAMP production. Apart from being desensitized by histamine, the internalization of the receptor was indistinguishable from that of the WT. Internalization was observed in the T-320 but not in T-313 mutant, narrowing 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 conserved 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. Furthermore, the finding that T-308 receptors were desensitized in the absence of internalization suggests that internalization and desensitization are meditated by independent mechanisms.