Goodman O B, Krupnick J G, Gurevich V V, Benovic J L, Keen J H
Department of Biochemistry, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 1997 Jun 6;272(23):15017-22. doi: 10.1074/jbc.272.23.15017.
Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.
此前我们证明，非视觉抑制蛋白与网格蛋白表现出高亲和力相互作用，这与它们在G蛋白偶联受体内化过程中的衔接蛋白功能一致（古德曼，O.B.，小克鲁普尼克，J.G.，桑蒂尼，F.，古列维奇，V.V.，佩恩，R.B.，加尼翁，A.W.，基恩，J.H.，以及贝诺维奇，J.L.（1996年）《自然》383卷，447 - 450页）。在本报告中我们表明，网格蛋白球状末端结构域内一段高度保守的短序列负责与抑制蛋白结合。对网格蛋白笼进行有限的蛋白酶解会导致末端结构域的释放以及随之而来的抑制蛋白结合能力的丧失。非视觉抑制蛋白β - 抑制蛋白和抑制蛋白3，但不是视觉抑制蛋白，能特异性结合谷胱甘肽S - 转移酶 - 网格蛋白末端结构域融合蛋白。缺失分析和丙氨酸扫描诱变将结合位点定位到网格蛋白重链的89 - 100位残基，并表明1 - 100位残基可作为一个独立的抑制蛋白结合结构域发挥作用。定点诱变确定一个不变的谷氨酰胺（Glu - 89）和两个高度保守的赖氨酸（Lys - 96和Lys - 98）是抑制蛋白结合的关键残基，这补充了抑制蛋白3中与网格蛋白结合有关的疏水和酸性残基（克鲁普尼克，J.G.，古德曼，O.B.，小，基恩，J.H.，以及贝诺维奇，J.L.（1997年）《生物化学杂志》272卷，15011 - 15016页）。尽管抑制蛋白表现出与网格蛋白的高亲和力结合，但它们不会诱导衣被组装。末端结构域在有被小窝中朝向质膜，它与抑制蛋白和AP - 2的结合表明该结构域是负责将衔接蛋白 - 受体招募到有被小窝的锚定结构域。
