Ferrari S L, Bisello A
Harvard-Thorndike and Charles A. Dana Research Laboratories, Department of Medicine Beth Israel Deaconess Medical Center and Harvard Medical School Boston, Massachusetts 02215, USA.
Mol Endocrinol. 2001 Jan;15(1):149-63. doi: 10.1210/mend.15.1.0587.
PTH promotes endocytosis of human PTH receptor 1 (PTH1Rc) by activating protein kinase C and recruiting beta-arrestin2. We examined the role of beta-arrestin2 in regulating the cellular distribution and cAMP signaling of two constitutively active PTH1Rc mutants, H223R and T410P. Overexpression of a beta-arrestin2-green fluorescent protein (GFP) conjugate in COS-7 cells inhibited constitutive cAMP accumulation by H223R and T410P in a dose-dependent manner, as well as the response to PTH of both mutant and wild-type PTH1Rcs. The cellular distribution of PTH1Rc-GFP conjugates, fluorescent ligands, and ssarrestin2-GFP was analyzed by fluorescence microscopy in HEK-293T cells. In cells expressing either receptor mutant, a ligand-independent mobilization of beta-arrestin2 to the cell membrane was observed. In the absence of ligand, H223R and wild-type PTH1Rcs were mainly localized on the cell membrane, whereas intracellular trafficking of T410P was also observed. While agonists promoted beta-arrestin2-mediated endocytosis of bot PTH1Rc mutants, antagonists were rapidly internalized only with T410P. The protein kinases inhibitor, staurosporine, significantly decreased internalization of ligand-PTH1Rc mutant complexes, although the recruitment of beta-arrestin2 to the cell membrane was unaffected. Moreover, in cells expressing a truncated wild-type PTH1Rc lacking the C-terminal cytoplasmic domain, agonists stimulated translocation of beta-arrestin2 to the cell membrane followed by ligand-receptor complex internalization without associated beta-arrestin2. In conclusion, cAMP signaling by constitutively active mutant and wild-type PTH1Rcs is inhibited by a receptor interaction with beta-arrestin2 on the cell membrane, possibly leading to uncoupling from G(s)alpha. This phenomenon is independent from protein kinases activity and the receptor C-terminal cytoplasmic domain. In addition, there are differences in the cellular localization and internalization features of constitutively active PTH1Rc mutants H223R and T410P.
甲状旁腺激素（PTH）通过激活蛋白激酶C并募集β-抑制蛋白2来促进人甲状旁腺激素受体1（PTH1Rc）的内吞作用。我们研究了β-抑制蛋白2在调节两种组成型活性PTH1Rc突变体H223R和T410P的细胞分布及cAMP信号传导中的作用。在COS-7细胞中过表达β-抑制蛋白2-绿色荧光蛋白（GFP）偶联物以剂量依赖性方式抑制了H223R和T410P的组成型cAMP积累，以及突变型和野生型PTH1Rc对PTH的反应。通过荧光显微镜在HEK-293T细胞中分析了PTH1Rc-GFP偶联物、荧光配体和β-抑制蛋白2-GFP的细胞分布。在表达任一受体突变体的细胞中，观察到β-抑制蛋白2向细胞膜的非配体依赖性动员。在无配体的情况下，H223R和野生型PTH1Rc主要定位于细胞膜，而T410P也存在细胞内转运。虽然激动剂促进了两种PTH1Rc突变体的β-抑制蛋白2介导的内吞作用，但拮抗剂仅与T410P一起迅速内化。蛋白激酶抑制剂星形孢菌素显著降低了配体-PTH1Rc突变体复合物的内化，尽管β-抑制蛋白2向细胞膜的募集未受影响。此外，在表达缺失C末端胞质结构域的截短野生型PTH1Rc的细胞中，激动剂刺激β-抑制蛋白2向细胞膜易位，随后配体-受体复合物内化且无相关的β-抑制蛋白2。总之，组成型活性突变体和野生型PTH1Rc的cAMP信号传导通过与细胞膜上的β-抑制蛋白2相互作用而受到抑制，这可能导致与G(s)α解偶联。这种现象独立于蛋白激酶活性和受体C末端胞质结构域。此外，组成型活性PTH1Rc突变体H223R和T410P在细胞定位和内化特征方面存在差异。
