A Novel Integrin Specificity Exemplified by Binding of the av / 35 Integrin to the Basic Domain of the HIV Tat Protein and Vitronectin-猫眼课题宝

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2002

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B. Vogel;Shuchen Lee;Axel Hi ldebrand;I. Craig;M. Pierschbacher;F. Wong-Staal;Erkki

一些研究已经解决了HIV Tat蛋白与细胞表面的相互作用。我们对Tat及其衍生肽的细胞附着促进活性的分析表明，Tat的基本结构域，而不是arg-gly-asp （RGD）序列，是细胞附着于Tat的必要条件。Tat肽的亲和层析和各种抗整合素抗体的免疫沉淀表明，外连接素结合整合素cxv/35是结合Tat基本结构域的细胞表面蛋白。Tat基本结构域包含序列RKKRRQRRR。一个相关的序列KKQRFRHRNRKG存在于aviS5配体玻璃连接蛋白的肝素结合区域，在亲和层析中也结合c~，/35，并且与RGD肽结合，是细胞粘附到玻璃连接蛋白的抑制剂。a~/35与这些肽的相互作用不仅仅是由于配体序列中碱性氨基酸的高含量；tXv/35不能与完全由精氨酸或赖氨酸组成的肽结合，而a/3t整合素可以与这些肽结合。c~v/35与Tat的相互作用对于整合素来说是不典型的，因为与Tat的结合是不依赖于二价阳离子的，而同一整合素与含有rgd的肽或玻璃体连接蛋白的结合则需要二价阳离子。这些数据定义了基本氨基酸序列的辅助整合素结合特异性。这些基本结构域结合位点可能与识别RGD或等效序列的结合位点协同作用。th ~三肽arg-gly-asp (RGD) 1是细胞粘附许多蛋白质所必需的，包括纤维连接蛋白、玻璃体连接蛋白和纤维蛋白原（Pierschbacher and Ruoslahti, 1984; Ruoslahti and Pierschbacher, 1987）。这种粘附是由整合素介导的，整合素是一个跨膜受体家族，由两个亚基组成，ot和/3 （Hemler, 1990; Ruoslahti, 1991; Hynes, 1992）。人类免疫缺陷病毒（HIV-1）的Tat蛋白含有一个RGD序列，可以以RGD依赖的方式介导细胞附着（Brake等，1990）。细胞外Tat被细胞内化并运输到细胞核，在那里它保留了反激活HIV启动子的能力（Frankel和Pabo， 1988）。此外，细胞外Tat已被证明可以调节细胞增殖，既可以抑制抗原活化的t细胞的增殖（visidi等人，1989），也可以刺激卡波西氏肉瘤（KS）衍生细胞的增殖（Ensoli等人，1990）。地址：拉霍亚癌症研究基金会Ruoslahti博士，地址：加州拉茹阿市北托里松路10901号，邮编：92037。沃格尔博士现在的地址是约翰霍普金斯大学生物系，3400 N. Charles Street, Baltimore, Maryland 21218。Hildebrand博士目前的地址是德国明斯特大学皮肤科，D-4400 Munster。本文使用的缩写：RGD， arg-gly-asp；HIV，人类免疫缺陷病毒。我们认为rgd结合整合素介导Tat与细胞表面的一些相互作用的可能性非常有趣，并着手鉴定这样的整合素。我们发现otvl3~整合素与Tat蛋白结合，但这种相互作用在细胞对Tat的摄取中并不显著。令人惊讶的是，我们的结果表明，这种整合素与Tat的结合需要基本区域，而RGD序列是沉默的。我们还提供证据表明，玻璃体连接蛋白的一个基本序列可以作为av/35整合素的结合位点，而整合素的结合需要一个合适的碱性氨基酸序列。这些结果表明，存在一种以前未被认识到的整合素特异性，指向由几种基本氨基酸组成的序列基序。材料与方法

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, cxv/35, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an aviS5 ligand, vitronectin, also bound c~,/35 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The a~/35 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; tXv/35 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a/3t integrin did bind to these peptides. The interaction of c~v/35 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD-containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences. T H~ tripeptide arg-gly-asp (RGD) 1 is required for cell adhesion to a number of proteins, including fibronectin, vitronectin, and fibrinogen (Pierschbacher and Ruoslahti, 1984; Ruoslahti and Pierschbacher, 1987). This adhesion is mediated by integrins, a family oftransmembrane receptors composed of two subunits, ot and/3 (Hemler, 1990; Ruoslahti, 1991; Hynes, 1992). The Tat protein of human immunodeficiency virus (HIV-1) contains an RGD sequence and can mediate cell attachment in an RGD-dependent manner (Brake et al., 1990). Extracellular Tat is internalized by cells and transported to the nucleus, where it retains the ability to transactivate the HIV promoter (Frankel and Pabo, 1988). Furthermore, extracellular Tat has been shown to modulate cell proliferation, both in the suppression of proliferation of antigen-activated T-cells (Viscidi et al., 1989) and in the stimulation of proliferation of Kaposi's sarcoma (KS)-derived cells (Ensoli et al., 1990). Address correspondence to Dr. Ruoslahti at La Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La JoUa, California 92037. Dr. Vogel's present address is Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, Maryland 21218. Dr. Hildebrand's present address is Department of Dermatology, University of Munster, D-4400 Munster, Germany. 1. Abbreviations used in this paper: RGD, arg-gly-asp; HIV, human immunodeliciency virus. We felt that the possibility of an RGD-binding integrin mediating some of the interactions of Tat with cell surfaces was of a considerable interest and set out to identify such an integrin. We found that the otvl3~ integrin bound to the Tat protein, but that this interaction was not significant in the uptake of Tat by cells. Surprisingly, our results indicate that the binding of this integrin to Tat requires the basic region, whereas the RGD sequence is silent. We also provide evidence that a basic sequence in vitronectin can serve as a binding site for the av/35 integrin and that the integrin binding requires an appropriate sequence of basic amino acids. These results suggest the existence of a previously unrecognized integrin specificity directed toward a sequence motif consisting of several basic amino acids. Materials and Methods

A Novel Integrin Specificity Exemplified by Binding of the av / 35 Integrin to the Basic Domain of the HIV Tat Protein and Vitronectin

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