Kusui T, Hellmich M R, Wang L H, Evans R L, Benya R V, Battey J F, Jensen R T
Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1804, USA.
Biochemistry. 1995 Jun 27;34(25):8061-75. doi: 10.1021/bi00025a012.
Whereas baculovirus expression systems have been extensively used for high-level expression of steroid receptors and receptors coupled to adenylate cyclase, there are few studies on peptide receptors coupled to phospholipase C (PLC). In the present study we have expressed the murine gastrin-releasing peptide receptor (mGRP-R) in Sf9 cells using a recombinant baculovirus and characterized it structurally and functionally. mGRP-R was detectible 12 h post infection with recombinant baculovirus carrying mGRP-R cDNA and became maximal at 60 h post infection (Bmax = 6 pmol/mg protein), which is a 4-60-fold greater density than is found in native tissues. The mGRP-R in Sf9 cells assessed by affinity labeling or immunoblotting was smaller than that in native tissues (M(r) = 51 kD vs 82 kD), and the difference was due to the extent of glycosylation. In Sf9 cells the mGRP-R had at least two of the four potential extracellular glycosylation sites glycosylated, whereas in the native receptor all four were approximately equally glycosylated. In Sf9 cells the glycosylation was entirely biantennary complex, in contrast to the native mGRP-R, where it was entirely tri- and tetraantennary complex N-linked oligosaccharides. Affinity labeling studies revealed a band with an apparent molecular mass about 40 kDa higher than the 51-kDa mGRP-R band. The intensity of this band correlated with the extent of functional G protein coupling, suggesting that it may represent an mGRP-R-G protein complex. In binding studies the affinity of the mGRP-r in Sf9 cells for the agonists bombesin (Bn), GRP, and neuromedin B (NMB) varied differently with infection time: with Bn the affinity decreased 3-fold with longer infection times, with GRP it remained unchanged, and with NMB it decreased 10-fold. GPP(NH)p inhibited binding of either [125I]Tyr4Bn or [125I]GRP at 24 h post infection, but not at 96 h post infection. Agonists activated PLC, increasing both [3H]IP and [Ca2+]i; however, the efficacy of each agonist decreased with infection time. These results demonstrate that by the use of recombinant baculovirus infected Sf9 cells the PLC-linked receptor mGRP-R can be expressed in amounts significantly greater than those in native tissues. The mGRP-R expressed in these Sf9 cells is incompletely glycosylated and has less complex N-linked oligosaccharide chains, yet it is fully coupled to G proteins and activates phospholipase C, similar to the native receptor, if short infection times are used.(ABSTRACT TRUNCATED AT 400 WORDS)
尽管杆状病毒表达系统已被广泛用于类固醇受体和与腺苷酸环化酶偶联的受体的高水平表达，但关于与磷脂酶C（PLC）偶联的肽受体的研究却很少。在本研究中，我们使用重组杆状病毒在Sf9细胞中表达了小鼠胃泌素释放肽受体（mGRP-R），并对其进行了结构和功能表征。用携带mGRP-R cDNA的重组杆状病毒感染后12小时可检测到mGRP-R，在感染后60小时达到最大值（Bmax = 6 pmol/mg蛋白），这一比天然组织中发现的密度高4至60倍。通过亲和标记或免疫印迹评估的Sf9细胞中的mGRP-R比天然组织中的小（相对分子质量M(r)=51 kD对82 kD），差异是由于糖基化程度不同。在Sf9细胞中，mGRP-R的四个潜在细胞外糖基化位点中至少有两个被糖基化，而在天然受体中，四个位点的糖基化程度大致相同。在Sf9细胞中，糖基化完全是双天线复合糖，而天然mGRP-R中的糖基化完全是三天线和四天线复合N-连接寡糖。亲和标记研究揭示了一条表观分子质量比51-kDa的mGRP-R条带高约40 kDa的条带。这条带的强度与功能性G蛋白偶联程度相关，表明它可能代表mGRP-R-G蛋白复合物。在结合研究中，Sf9细胞中mGRP-r对激动剂蛙皮素（Bn）、GRP和神经介素B（NMB）的亲和力随感染时间的变化不同：对于Bn，亲和力随感染时间延长降低3倍，对于GRP保持不变，对于NMB降低10倍。GPP(NH)p在感染后24小时抑制[125I]Tyr4Bn或[125I]GRP的结合，但在感染后96小时不抑制。激动剂激活PLC，增加[3H]IP和[Ca2+]i；然而，每种激动剂的效力随感染时间降低。这些结果表明，通过使用重组杆状病毒感染的Sf9细胞，与PLC偶联的受体mGRP-R可以以比天然组织中显著更高的量表达。在这些Sf9细胞中表达的mGRP-R糖基化不完全，具有较不复杂的N-连接寡糖链，但如果使用较短的感染时间，它与G蛋白完全偶联并激活磷脂酶C，类似于天然受体。（摘要截短至400字）
