Bommakanti R K, Klotz K N, Dratz E A, Jesaitis A J
Department of Microbiology, Montana State University, Bozeman 59717.
J Leukoc Biol. 1993 Dec;54(6):572-7. doi: 10.1002/jlb.54.6.572.
The binding of G protein to the N-formyl peptide receptor of human neutrophils was investigated with site-specific synthetic peptides. Peptide CT336(322) (322RALTEDSTQTSDTAT336) from the carboxyl-terminal tail region of the receptor competed with the receptor for binding to bovine Gi protein. The peptide competition was assayed by dissociation of a GTP-sensitive, rapidly sedimenting (7S) form of receptor-G protein complex as analyzed by velocity sedimentation on linear sucrose density gradients. An IC50 of 590 microM was determined for CT336(322) peptide. A control peptide, with the reverse sequence, rCT322(336) (336TATDSTQTSDETLAR322), did not perturb the sedimentation of the reconstituted receptor-G protein complex up to the highest tested concentration, 3 mM. Other peptides tested, corresponding to central portions of the predicted intracellular loop regions CII140(127) (127VLHPVWTQNHRTVS140) and CIII239(227) (227KIHKQGLIKSSRP239) of the receptor, failed to dissociate the reconstituted receptor-G protein complex. Control peptides from the extracellular region EII184(170) (170KTGTVACTFNFSPWT184) and an unrelated sequence matching a portion of neutrophil cytochrome b, CYT306(296) (296KVVITKVVTHPFKTIE306), were also ineffective. Our results suggest that the cytoplasmic tail of the formyl chemotactic peptide receptor is involved in its coupling to the signal-transducing G protein.
利用位点特异性合成肽研究了G蛋白与人中性粒细胞N-甲酰基肽受体的结合。来自受体羧基末端尾部区域的肽CT336(322)（322RALTEDSTQTSDTAT336）与受体竞争结合牛Gi蛋白。通过在线性蔗糖密度梯度上进行速度沉降分析，以解离GTP敏感的、快速沉降（7S）形式的受体-G蛋白复合物来测定肽竞争。测定CT336(322)肽的IC50为590 microM。具有反向序列的对照肽rCT322(336)（336TATDSTQTSDETLAR322）在高达3 mM的最高测试浓度下均未干扰重组受体-G蛋白复合物的沉降。测试的其他肽，对应于受体预测的细胞内环区域CII140(127)（127VLHPVWTQNHRTVS140）和CIII239(227)（227KIHKQGLIKSSRP239）的中央部分，未能解离重组受体-G蛋白复合物。来自细胞外区域EII184(170)（170KTGTVACTFNFSPWT184）的对照肽以及与中性粒细胞细胞色素b的一部分匹配的无关序列CYT306(296)（296KVVITKVVTHPFKTIE306）也无效。我们的结果表明，甲酰基趋化肽受体的细胞质尾部参与其与信号转导G蛋白的偶联。
