Role of the C Terminus in Histamine H2 Receptor Signaling, Desensitization, and Agonist-induced Internalization

Abstract

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 cellsurface expression per cell was equivalent. As observed in a number of guanine nucleotide-binding protein-coupled receptors (GPCRs), 1 cAMP responses occurring via the histamine H2 receptor are rapidly desensitized after agonist stimulation (1-5). The H2 receptor also exhibits an agonist-induced internalization from the cell surface (2, 6). However, the mechanisms underlying these phenomena have yet to be identified. Recently, a number of studies focusing on GPCRs have examined the role of the C terminus in agonistinduced desensitization and internalization, with varying results. For example, C-terminal truncation of the angiotensin II receptor inhibited agonist-induced internalization of this receptor, but not desensitization of the calcium response mediated via the receptor (7). On the other hand, C-terminal truncation of ␤ 2 -adrenergic, ␣ 1B -adrenergic, lutropin/choriogonadotropin, platelet-activating factor, and neurokinin-2 receptors resulted in impairment of homologous desensitization (8 -12). Thus, the C termini of GPCRs have functional importances, which may differ among receptors. The present study was designed to analyze the role of this important portion of the histamine H2 receptor in signaling, desensitization, and agonist-induced internalization. To this end, we constructed H2 receptor cDNAs, devoid of either the 71 or the 51 amino acids at the C terminus, and expressed these cDNAs in COS7 cells. Herein, we present evidence that the 51 C-terminal amino acids of the histamine H2 receptor are important for agonist-induced internalization, but not for either signaling or homologous desensitization of cAMP response, which occurs via this receptor. In addition, these amino acids exert inhibitory effects on cAMP production via the H2 receptor.

Recent Advances in Molecular Pharmacology of the Histamine Systems: Roles of C-terminal Tails of Histamine Receptors

“…Truncations or replacements of amino acids in the C-terminal region of the receptor revealed that Thr 315 is involved in agonistinduced internalization. Interestingly, the truncated H 2 receptor mutant that deletes Thr 315 still showed desensitization measured by the cAMP production, suggesting that internalization and desensitization are mediated by independent mechanisms (19). Similar results were shown in CHO cells stably transfected with rat H 2 receptor (20).…”

“…Similar to many other GPCRs, histamine H 2 receptor is desensitized and internalized after agonist stimulation. In COS-7 cells transiently transfected with canine histamine H 2 receptor cDNA, the amount of receptors on the cell surface decreased by approximately 30% after the treatment with 10 −5 M histamine, while total amount of receptor measured by [ 3 H]tiotidine binding did not change, suggesting the internalization of the receptors from the cell surface (19). Truncations or replacements of amino acids in the C-terminal region of the receptor revealed that Thr 315 is involved in agonistinduced internalization.…”

“…When the most of the C-terminal tail was removed from canine H 2 receptor, which also deletes the Cys 305 residue, a possible palmitoylation site (Fig. 3), the truncated receptor expressed in COS-7 cells showed mainly intracellular distribution with less amount of surface receptor (19). Because the distribution to the plasma membrane is preserved in the receptor lacking 51 amino acids in the C-terminal tail, the Nterminal portion of the C-terminus, including the palmitoylation of Cys 305 seems to have an important role in the membrane targeting of the H 2 receptor (19).…”

“…3), the truncated receptor expressed in COS-7 cells showed mainly intracellular distribution with less amount of surface receptor (19). Because the distribution to the plasma membrane is preserved in the receptor lacking 51 amino acids in the C-terminal tail, the Nterminal portion of the C-terminus, including the palmitoylation of Cys 305 seems to have an important role in the membrane targeting of the H 2 receptor (19). There has been no report so far regarding the functional roles for the carboxy-termini of other histamine receptors, that is, H 1 , H 3 , and H 4 receptors.…”

Molecular characterisation of the adiponectin receptors, AdipoR1 and AdipoR2

“…Deletion of this entire C-terminal tail in the histamine H2 receptor demonstrated a potential role in determining cell surface expression 130 while in the angiotensin II and the Bradykinin B2 receptors, C-terminal truncations did not affect cell surface expression but compromised the internalisation of each receptor 131,132 . The C-terminal tail has also been implicated in agonist-induced receptor phosphorylation in GPCRs such as the delta opioid receptor and the α1B-Adrenergic receptor 133,134 and may also function in agonist induced receptor desensitisation as shown for the α1B-Adrenergic receptor 133 .…”