Identification of Cellular Receptors Involved in HIV Infection, Tumor Metastasis

HIV-1包膜蛋白的两个组分gp120和gp41被发现能有效地抑制人单核细胞上趋化受体的表达和功能，包括趋化因子受体和细菌趋化肽FMLF的受体。HIV-1被膜蛋白的抑制作用不是通过与其他趋化物质直接竞争受体，而是通过一种涉及蛋白激酶C激活的“异源脱敏”机制实现的。gp120还抑制人CD4T淋巴细胞趋化因子受体CXCR4的表达和功能，这需要酪氨酸激酶(P56lck)激活所介导的信号事件。我们进一步研究了人类白细胞与HIV-1包膜蛋白中在HIV-1融合中起重要作用的多肽结构域之间的相互作用，发现人吞噬细胞上的两个跨膜受体，即FPRR和FPRL1，可被一些HIV-1包膜结构域激活。这些受体最初被认为是趋化性N-甲酰化多肽的激活部位，这些多肽可以是合成的，也可以是由某些细菌和细胞质线粒体产生的。这些观察结果表明，虽然HIV-1包膜蛋白在介导病毒入侵中起关键作用，但它们的结构域也可能诱导宿主免疫反应，可能导致宿主天然免疫反应的激活。然而，免疫细胞的有效和长期的激活也可能导致细胞对其他刺激物的无反应，这是对艾滋病相关的机会性感染有利的脱敏的结果。由于趋化因子受体CCR5和CXCR4是人类细胞感染HIV-1的关键辅助受体，我们探索了利用受体脱敏作为开发新型抗HIV-1药物的可能性。我们发现，细菌FMLF激活FPR之后，蛋白激酶C介导的CCR5的丝氨酸磷酸化与CCR5的减弱有关，包括它作为HIV-1辅助受体的能力。我们还鉴定了一种随机合成的多肽，W肽，它既能激活FPRL1，又能激活FPRR，并能抑制HIV-1对受体转基因细胞系和人外周血单核细胞的感染。W肽序列中含有D-氨基酸，可抵抗酶降解，更适合于体内给药以检测其抗HIV-1的效果。血清淀粉样蛋白A(SAA)是一种急性时相蛋白，通常存在于血清中0.1 mM的水平，但在全身炎症条件下会增加1000倍。血清SAA升高的慢性炎症状态可能最终导致淀粉样变性，其特征是组织中“淀粉样蛋白”纤维的沉积，并与器官功能的进行性破坏有关。我们发现，重组人(Rh)SAA在体外对人白细胞具有相当大的趋化活性，并诱导这些细胞在小鼠体内的注射部位渗透，这表明当SAA局部存在时，可能起到促炎作用。为了确定SAA的细胞受体，我们仔细评估了SAA诱导的细胞信号模式和其他趋化因子的交叉脱敏。我们发现高浓度的细菌衍生趋化肽fMLP能够抑制细胞对SAA刺激的反应，提示SAA可能使用与fMLP低亲和力的白细胞受体。通过利用基因工程技术表达编码各种七种跨膜G蛋白偶联受体的基因，我们鉴定了一种称为FPRL1的受体，该受体可被SAA特异性结合并激活。SAA受体的鉴定将为进一步研究SAA在急性期反应和组织淀粉样变性中的作用奠定基础。

Two components of the HIV-1 envelope protein, gp120 and gp41, were found to potently inhibit the expression and function of chemoattractant receptors on human monocytes, including the receptors for chemokines as well as for bacterial chemotactic peptide fMLF. The inhibitory effects of HIV-1 envelope proteins was not due to a direct competition for the receptors with other chemoattractants, but rather, through a "heterologous desensitization" mechanism involving the activation of protein kinase C. Gp120 also inhibited the expression and function of chemokine receptor CXCR4 in human CD4+ T lymphocytes, and this required signaling events mediated by tyrosine kinase(p56lck) activation. We further investigated the interaction of human leukocytes with peptide domains in HIV-1 envelope proteins that are important in HIV-1 fusion and found that two 7 transmembrane receptors on human phagocytes, FPR and FPRL1, are activated by some HIV-1 envelope domains. These receptors were originally identified as activation sites for chemotactic N-formylated peptides, either synthetic or produced by certain bacteria and cytoplasmic mitochondria. These observations suggest that while HIV-1 envelope proteins are crucial in mediating viral invasion, their domains may also elicit host immune responses possibly resulting in activation of host innate immune responses. However, a potent and prolonged activation of immune cells may alsoculminate in unresponsiveness of the cells to other stimulants, a consequence of "desensitization" which could be beneficial for AIDS-related opportunistic infection. Since the chemokine receptors CCR5 and CXCR4 are crucial co-receptors of HIV-1 infection of human cells, we investigated the possibility to utilize receptor desensitization as a means of develop novel anti-HIV-1 agents. We have revealed that activation of FPR by the bacterial fMLF was followed by a rapid serine phosphorylation of CCR5 mediated by protein kinase C, in association with the attenuation of CCR5 including its capacity to act as an HIV-1 coreceptor. We also identified a random synthetic peptide, W peptide, that activates both FPR and FPRL1 and inhibits HIV-1 infection of receptor transfected cell lines as well as human peripheral blood mononuclear cells. W peptide contains a D-amino acid in its sequence and thus may be resistant to enzymatic degradation and more suitable for in vivo administration to test its anti-HIV-1 efficacy. Serum amyloid A (SAA), an acute phase protein, is normally present in serum at 0.1 mM levels, but increases by 1000 fold in systemic inflammatory conditions. Chronic inflammatory conditions with elevated serum SAA may culminate in amyloidosis, characterized by deposition of "amyloid" fibrils in tissues and associated with progressive destruction of organ function. We found that recombinant human (rh) SAA exhibited considerable chemoattractant activity for human leukocytes in vitro and induced infiltration of these cells at injection sites in mice, suggesting that SAA, when present locally, may play a proinflammatory role. In an effort to identify the cell receptor for SAA, we carefully evaluated the pattern of cell signaling induced by SAA and cross-desensitization by other chemotactic factors. We found that the bacterial derived chemotactic peptide fMLP at high concentrations was able to inhibit the cell response to subsequent stimulation with SAA, suggesting that SAA might use a leukocyte receptor for which fMLP has low affinity. By using cell lines engineered to express genes encoding various seven-transmembrane, G-protein coupled receptors, we identified a receptor termed FPRL1 to be specifically bound and activated by SAA. The identification of the receptor used by SAA will greatly facilitate the further research on the role of SAA in acute phase responses and tissue amyloidosis.