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 T³⁰⁸ mutant) without loss of functions: cAMp production, tiotidine binding, and plasma membrane targeting. In fact, the T³⁰⁸ mutant produced more cAMP than the WT when cell-surface expression per cell was equivalent. Pretreatment of cells with 10^-5 M histamine desensitized cAMP productions via WT and T³⁰⁸ receptors to 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) ~ 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 T³⁰⁸ receptors following histamine treatment. A mutant with a deletion of the 30 C-terminal amino acids, termed T³²⁹, 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 T³²⁰ but not in T³¹³ mutant, narrowing the region involved in internalization to that between Glu³¹⁴ and Asn³²⁰ (ETSLRSN). Of these seven residues, either Thr³¹⁵, Ser³¹⁶, or both, were replaced with Ala. Thr³¹⁵ and Ser³¹⁶ are conserved among species. The mutation at Thr³¹⁵ (but not that at Ser³¹⁶) abolished internalization. Taken together, these results demonstrate that Thr³¹⁵ is involved in agonist-induced internalization. Furthermore, the finding that T³⁰⁸ receptors were desensitized in the absence of internalization suggests that internalization and desensitization are meditated by independent mechanisms.