TY - JOUR
T1 - Role of the C terminus in histamine H2 receptor signaling, desensitization, and agonist-induced internalization
AU - Fukushima, Yasushi
AU - Asano, Tomoichiro
AU - Takata, Kuniaki
AU - Funaki, Makoto
AU - Ogihara, Takehide
AU - Anai, Motonobu
AU - Tsukuda, Katsunori
AU - Saitoh, Toshihito
AU - Katagiri, Hideki
AU - Aihara, Makoto
AU - Matsuhashi, Nobuyuki
AU - Oka, Yoshitomo
AU - Yazaki, Yoshio
AU - Sugano, Kentaro
PY - 1997/8/1
Y1 - 1997/8/1
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.272.31.19464
DO - 10.1074/jbc.272.31.19464
M3 - Article
C2 - 9235948
AN - SCOPUS:0030840733
VL - 272
SP - 19464
EP - 19470
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 31
ER -