Recombinant Human CXC-Chemokine Receptor-4 in Melanophores Are Linked to G_iProtein: Seven Transmembrane Coreceptors for Human Immunodeficiency Virus Entry into Cells

Abstract

This article describes the transient expression of the CXC chemokine receptor-4 in Xenopus laevis melanophores and the resulting functional assay for the endogenous ligand for this receptor stromal cell-derived factor (SDF)-1␣. Specifically, it will be shown that SDF-1␣ produces increased light transmittance in transfected cells that is consistent with the activation of G i protein. This stimulus pathway is further implicated by the abolition of this response after pretreatment of the cells with pertussis toxin, a known method for the inactivation of G i protein. The fact that SDF-1␣ does not produce responses in nontransfected cells and that treatment of the cells with 12G5, an antibody specific for the CXC chemokine receptor-4, eliminates this response indicates that this ligand produces responses by activation of this receptor in these cells. The possible relevance to human immunodeficiency virus (HIV) entry into cells was explored by observing the effects of SDF-1␣ on HIV-mediated cell fusion. It was found that SDF-1␣ blocked cell-to-cell fusion (as has been previously reported) at concentrations 1200-fold greater than those required to produce G i protein mediated responses. The implications of the functional assay to screening for new drugs to block HIV-mediated fusion is discussed.The discovery that HIV-1 utilizes seven transmembrane receptors as coreceptors for viral fusion has introduced a new target into the therapeutic field of view. Studies on HIV-1 envelope-mediated cell fusion have identified a 46-kDa, integral membrane protein named "fusin" that serves as a cofactor for HIV fusion and entry (Feng et al., 1996). Fusin is a 352-amino-acid, seven-transmembrane receptor, the sequence of which is identical to a previously cloned orphan receptor denoted LESTR (leukocyte-derived seven-transmembrane domain receptor). Sequence comparison has shown that fusin (or LESTR) is a member of the CXC chemokine receptor family with a sequence homology to the CXCR2 receptor (Loetscher et al., 1994;Raport et al., 1996;Wells et al., 1996). The identification of SDF-1␣ (a CXC chemokine) as a ligand for fusin has led to the suggested classification of this receptor as CXCR4 in the chemokine receptor nomenclature system (Bleul et al., 1996;Oberlin et al., 1996). This article describes the construction of a receptor assay in which the human CXCR4 couples to G i -protein in recombinant Xenopus laevis melanophores. In these cells, melanosome dispersion can be affected via activation of adenylyl cyclase (Potenza et al., 1992;McClintock et al., 1993) or phospholipase C , whereas melanosome aggregation results from the inhibition of adenylyl cyclase (Potenza et al., 1992;McClintock et al., 1993). Melanophore cells contain a wide range of G ␣ -proteins (Jayawickreme et al., 1994); therefore, the expression of numerous foreign G protein-coupled receptors can be facilitated (Potenza et al., 1992(Potenza et al., , 1994Karne et al., 1993;McClintock et al., 1993;Graminski et al., 1993 and1994;Jayawickreme et al., 1994aJayawic...

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