Dynamic regulation of a GPCR-tetraspanin-G protein complex on intact cells:: Central role of CD81 in facilitating GPR56-Gαq/11 association
Dynamic regulation of a GPCR-tetraspanin-G protein complex on intact cells:: Central role of CD81 in facilitating GPR56-Gαq/11 association
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DOI：
10.1091/mbc.e03-12-0886
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发表时间：
2004-05-01
影响因子：
3.3
通讯作者：
Stipp, CS
中科院分区：
文献类型：
--
作者：
Little, KD;Hemler, ME;Stipp, CS
大量的异三聚体G蛋白偶联受体(GPCRs)通过多种细胞内支架蛋白，通过数量少得多的异三聚体G蛋白实现信号的特异性。Tetraspanin家族的成员组织了大量的细胞表面蛋白复合体，因此有可能作为GPCR支架；然而，Tetraspanin-GPCR复合体以前没有被描述过。我们现在发现GPCRGPR56/TM7XN1和异源三聚体G蛋白亚基Galpha(Q)、Galpha(Q)和Gbeta特异性地与Tetraspanins CD9和CD81结合，但不与其他Tetraspanins结合。GPR56与CD9和CD81的复合物在完全溶解时保持完好，并能抵抗胆固醇的消耗。因此，它们的稳定性不依赖于洗涤剂--不溶于水的、类筏状的膜微区。CD81免疫耗竭和再表达实验揭示了CD81在促进或稳定GPR56-CD81-Galpha(Q/11)复合体中的核心作用。最后，细胞表面CD81的抗体结合或细胞与佛波酯的激活揭示了GPR56-CD81-Galpha(Q/11)复合体动态调节的两种不同机制。这些数据揭示了Tetraspanins CD9和CD81作为GPCR支架蛋白的潜在作用。
By means of a variety of intracellular scaffolding proteins, a vast number of heterotrimeric G protein-coupled receptors (GPCRs) may achieve specificity in signaling through a much smaller number of heterotrimeric G proteins. Members of the tetraspanin family organize extensive complexes of cell surface proteins and thus have the potential to act as GPCR scaffolds; however, tetraspanin-GPCR complexes had not previously been described. We now show that a GPCR, GPR56/TM7XN1, and heterotrimeric G protein subunits, Galpha(q), Galpha(q), and Gbeta, associate specifically with tetraspanins CD9 and CD81, but not with other tetraspanins. Complexes of GPR56 with CD9 and CD81 remained intact when fully solubilized and were resistant to cholesterol depletion. Hence they do not depend on detergent-insoluble, raft-like membrane microdomains for stability. A central role for CD81 in promoting or stabilizing a GPR56-CD81-Galpha(q/11) complex was revealed by CD81 immunodepletion and reexpression experiments. Finally, antibody engagement of cell surface CD81 or cell activation with phorbol ester revealed two distinct mechanisms by which GPR56-CD81-Galpha(q/11) complexes can be dynamically regulated. These data reveal a potential role for tetraspanins CD9 and CD81 as GPCR scaffolding proteins.