Orsini M J, Benovic J L
Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 1998 Dec 18;273(51):34616-22. doi: 10.1074/jbc.273.51.34616.
Arrestins have been shown to act as adaptor proteins that mediate the interaction of G protein-coupled receptors with the endocytic machinery. In this study, the role of arrestin-3 in receptor internalization was investigated by constructing different arrestin-3 minigenes that could potentially act as dominant negative inhibitors of arrestin function. Expression of arrestin-3 proteins containing amino acids 1-320 or 201-409 resulted in the inhibition of beta2-adrenergic receptor internalization in HEK-293 cells by approximately 40%. Both of these arrestins were diffusely localized within the cytoplasm of transfected cells, were unable to mediate redistribution of receptors to clathrin-coated pits, and did not localize to coated pits in either the presence or absence of receptor and agonist. Arrestin-3(1-320), but not arrestin-3(201-409), bound to light-activated phosphorylated rhodopsin with an affinity comparable with that of wild-type arrestin-3. In contrast, expression of arrestin-3 proteins composed of only the clathrin binding domain, arrestin-3(284-409), and arrestin-3(290-409) resulted in the constitutive localization of these arrestins to coated pits. Arrestin-3(284-409) and arrestin-3(290-409) acted as dominant negative inhibitors of wild-type arrestin function, inhibiting receptor internalization by 70 and 30%, respectively. Carboxyl-terminal deletions of arrestin-3 retained the ability to promote internalization until residues amino-terminal to amino acid 350 were deleted, suggesting that residues in this region also compose part of the clathrin binding domain in addition to the major binding site between residues 371-379. These studies characterize at least two distinct mechanisms, competition for either receptor or clathrin binding, by which dominant negative arrestins inhibit receptor internalization and further define residues within arrestin-3 that constitute the clathrin binding domain.
抑制蛋白已被证明可作为衔接蛋白，介导G蛋白偶联受体与内吞机制的相互作用。在本研究中，通过构建不同的抑制蛋白-3小基因来研究抑制蛋白-3在受体内化中的作用，这些小基因可能作为抑制蛋白功能的显性负性抑制剂。含有氨基酸1-320或201-409的抑制蛋白-3蛋白的表达导致HEK-293细胞中β2-肾上腺素能受体内化受到约40%的抑制。这两种抑制蛋白均弥散分布于转染细胞的细胞质中，无法介导受体重新分布至网格蛋白包被小窝，且在有或无受体及激动剂的情况下均不定位至包被小窝。抑制蛋白-3(1-320)，而非抑制蛋白-3(201-409)，与光激活的磷酸化视紫红质结合，其亲和力与野生型抑制蛋白-3相当。相反，仅由网格蛋白结合结构域组成的抑制蛋白-3蛋白，即抑制蛋白-3(284-409)和抑制蛋白-3(290-409)的表达导致这些抑制蛋白组成型定位至包被小窝。抑制蛋白-3(284-409)和抑制蛋白-3(290-409)作为野生型抑制蛋白功能的显性负性抑制剂，分别抑制受体内化70%和30%。抑制蛋白-3的羧基末端缺失保留了促进内化的能力，直到氨基酸350氨基末端的残基被缺失，这表明该区域的残基除了371-379残基之间的主要结合位点外，也构成了网格蛋白结合结构域的一部分。这些研究描述了至少两种不同的机制，即对受体或网格蛋白结合的竞争，显性负性抑制蛋白通过这些机制抑制受体内化，并进一步确定了抑制蛋白-3内构成网格蛋白结合结构域的残基。
