Petrou C, Chen L, Tashjian A H
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
J Biol Chem. 1997 Jan 24;272(4):2326-33. doi: 10.1074/jbc.272.4.2326.
To determine whether functional receptor-G protein coupling or signaling are required for internalization of the thyrotropin-releasing hormone receptor (TRHR), we compared the endocytosis of Gq-coupled and uncoupled receptors. A hemagglutinin epitope-tagged TRHR (HA-TRHR) was in the Gq-coupled state when bound to the agonist, MeTRH, and in a nonsignaling state when bound to the HA antibody (12CA5). 12CA5 did not induce an increase in [Ca2+]i or inositol phosphates and did not inhibit [3H]MeTRH binding or MeTRH-induced production of second messengers. Both agonist- and antibody-bound HA-TRHRs were rapidly internalized via the same pathway; internalization was sensitive to hypertonic shock, and both types of internalized receptors were sorted into lysosomes. In addition, the amino acid sequence CNC (positions 335-337) in the C-terminal tail of the TRHR, which is important in ligand-induced receptor internalization as determined by deletion mutagenesis (Nussenzveig, D. R., Heinflink, M., and Gershengorn, M. C. (1993) J. Biol. Chem. 268, 2389-2392), was also important for 12CA5-induced internalization. We expressed two truncated receptors, HA-K338STOP and HA-C335STOP, in GH12C1 pituitary cells. Both HA-TRHR and HA-K338STOP were localized at the plasma membrane of untreated cells and were translocated to intracellular vesicles after MeTRH or 12CA5 binding; however, HA-C335STOP was internalized and recycled constitutively. The intracellular localization of HA-C335STOP was not altered by MeTRH; however, 12CA5 binding induced the disappearance of internalized HA-C335STOP and caused its localization at the plasma membrane, indicating that constitutively cycling HA-C335STOP cannot be reinternalized after antibody binding. Thus, amino acids 335-337, which are important for the internalization of Gq-coupled TRHRs, are also required for the sequestration of functionally uncoupled TRHRs, and in addition, they act as an inhibitory signal that prevents constitutive receptor internalization. Specifically, the Cys residues at positions 335 and 337 are important for preventing constitutive TRHR internalization, because a mutant HA-C335S/C337S receptor was sequestered constitutively. We conclude that release from a negative regulatory internalization sequence or domain is important for HA-TRHR internalization and that the role of the CNC sequence in internalization is independent of functional TRHR-Gq coupling.
为了确定促甲状腺激素释放激素受体（TRHR）内化是否需要功能性受体 - G蛋白偶联或信号传导，我们比较了与Gq偶联和未偶联受体的内吞作用。当血凝素表位标记的TRHR（HA - TRHR）与激动剂MeTRH结合时处于Gq偶联状态，而与HA抗体（12CA5）结合时处于无信号传导状态。12CA5不会诱导细胞内钙离子浓度（[Ca2+]i）或肌醇磷酸增加，也不会抑制[3H]MeTRH结合或MeTRH诱导的第二信使产生。与激动剂和抗体结合的HA - TRHRs都通过相同途径快速内化；内化对高渗休克敏感，并且两种类型的内化受体都被分选到溶酶体中。此外，TRHR C末端尾巴中的氨基酸序列CNC（第335 - 337位）在配体诱导的受体内化中很重要（通过缺失诱变确定，Nussenzveig, D. R., Heinflink, M., and Gershengorn, M. C. (1993) J. Biol. Chem. 268, 2389 - 2392），对于12CA5诱导的内化也很重要。我们在GH12C1垂体细胞中表达了两种截短受体，HA - K338STOP和HA - C335STOP。HA - TRHR和HA - K338STOP在未处理细胞的质膜上定位，在MeTRH或12CA5结合后转移到细胞内小泡中；然而，HA - C335STOP组成性内化并循环。MeTRH不会改变HA - C335STOP的细胞内定位；但是，12CA5结合会导致内化的HA - C335STOP消失并使其定位在质膜上，表明组成性循环的HA - C335STOP在抗体结合后不能再内化。因此，对于与Gq偶联的TRHR内化很重要的第335 - 337位氨基酸，对于功能未偶联的TRHR的隔离也是必需的，此外，它们还作为一种抑制信号，防止受体组成性内化。具体而言，第335和337位的半胱氨酸残基对于防止TRHR组成性内化很重要，因为突变体HA - C335S/C337S受体组成性地被隔离。我们得出结论，从负调节内化序列或结构域释放对于HA - TRHR内化很重要，并且CNC序列在内化中的作用独立于功能性TRHR - Gq偶联。
