Krasnoperov V, Bittner M A, Holz R W, Chepurny O, Petrenko A G
Departments of Pharmacology, New York University Medical Center, New York, New York 10016, USA.
J Biol Chem. 1999 Feb 5;274(6):3590-6. doi: 10.1074/jbc.274.6.3590.
Stimulation of neurotransmitter release by alpha-latrotoxin requires its binding to the calcium-independent receptor of alpha-latrotoxin (CIRL), an orphan neuronal G protein-coupled receptor. CIRL consists of two noncovalently bound subunits, p85, a heptahelical integral membrane protein, and p120, a large extracellular polypeptide with domains homologous to lectin, olfactomedin, mucin, the secretin receptor family, and a novel structural motif common for large orphan G protein-coupled receptors. The analysis of CIRL deletion mutants indicates that the high affinity alpha-latrotoxin-binding site is located within residues 467-891, which comprise the first transmembrane segment of p85 and the C-terminal half of p120. The N-terminal lectin, olfactomedin, and mucin domains of p120 are not required for the interaction with alpha-latrotoxin. Soluble p120 and all its fragments, which include the 467-770 residues, bind alpha-latrotoxin with low affinity suggesting the importance of membrane-embedded p85 for the stabilization of the complex of the toxin with p120. Two COOH-terminal deletion mutants of CIRL, one with the truncated cytoplasmic domain and the other with only one transmembrane segment left of seven, supported both alpha-latrotoxin-induced calcium uptake in HEK293 cells and alpha-latrotoxin-stimulated secretion when expressed in chromaffin cells, although with a different dose dependence than wild-type CIRL and its N-terminal deletion mutant. Thus the signaling domains of CIRL are not critically important for the stimulation of exocytosis in intact chromaffin cells by alpha-latrotoxin.
α- 银环蛇毒素刺激神经递质释放需要其与α- 银环蛇毒素的钙非依赖性受体（CIRL）结合，CIRL是一种孤儿神经元G蛋白偶联受体。CIRL由两个非共价结合的亚基组成，p85是一种七螺旋整合膜蛋白，p120是一种大型细胞外多肽，其结构域与凝集素、嗅觉介质、粘蛋白、促胰液素受体家族以及大型孤儿G蛋白偶联受体共有的新型结构基序同源。对CIRL缺失突变体的分析表明，高亲和力α- 银环蛇毒素结合位点位于467 - 891位残基内，该区域包括p85的第一个跨膜片段和p120的C端一半。p120的N端凝集素、嗅觉介质和粘蛋白结构域对于与α- 银环蛇毒素的相互作用不是必需的。可溶性p120及其所有片段（包括467 - 770位残基）以低亲和力结合α- 银环蛇毒素，这表明膜嵌入的p85对于毒素与p120复合物的稳定很重要。CIRL的两个COOH端缺失突变体，一个具有截短的胞质结构域，另一个仅剩下七个跨膜片段中的一个，在HEK293细胞中支持α- 银环蛇毒素诱导的钙摄取，在嗜铬细胞中表达时支持α- 银环蛇毒素刺激的分泌，尽管其剂量依赖性与野生型CIRL及其N端缺失突变体不同。因此，CIRL的信号结构域对于α- 银环蛇毒素在完整嗜铬细胞中刺激胞吐作用并非至关重要。
