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

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