Richardson R M, Ali H, Tomhave E D, Haribabu B, Snyderman R
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1995 Nov 17;270(46):27829-33. doi: 10.1074/jbc.270.46.27829.
To define the molecular mechanisms of cross-regulation among chemoattractant receptors, we stably coexpressed, in a rat basophilic leukemia (RBL-2H3) cell line, epitope-tagged receptors for the chemoattractants formylmethionylleucylphenylalanine (fMLP), a peptide of the fifth component of the complement system (C5a), and interleukin-8 (IL-8). All the expressed receptors underwent homologous phosphorylation and desensitization upon agonist stimulation. When co-expressed, epitope-tagged C5a receptor (ET-C5aR) and epitope-tagged IL-8 receptor (ET-IL-8RA) were cross-phosphorylated by activation of the other. Activation of epitope-tagged fMLP receptor (ET-FR) also cross-phosphorylated ET-C5aR and ET-IL-8RA, but ET-FR was totally resistant to cross-phosphorylation. Similarly, C5a and IL-8 stimulation of [35S]guanosine 5'-3-O-(thio) triphosphate (GTP gamma S) binding and Ca2+ mobilization were cross-desensitized by each other and by fMLP. Stimulation of [35S]GTP gamma S binding by fMLP was also not cross-desensitized by C5a or IL-8, however, Ca2+ mobilization was, suggesting a site of inhibition distal to G protein activation. Consistent with this desensitization of Ca2+ mobilization, inositol 1,4,5-trisphosphate release in RBL-2H3 cells expressing both ET-C5aR and ET-FR revealed that fMLP and C5a cross-desensitized each other's ability to stimulate phosphoinositide hydrolysis. Taken together, these results indicate that receptor cross-phosphorylation correlates directly with desensitization at the level of G protein activation. The ET-FR was resistant to this process. Of note, cross-desensitization of ET-FR at the level of phosphoinositide hydrolysis and Ca2+ mobilization was demonstrated in the absence of receptor phosphorylation. This suggests a new form of chemoattractant cross-regulation at a site distal to receptor/G protein coupling, involving the activity of phospholipase C.
为了确定趋化因子受体之间交叉调节的分子机制，我们在大鼠嗜碱性白血病（RBL-2H3）细胞系中稳定共表达了带有表位标签的趋化因子受体，这些趋化因子包括甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）、补体系统第五成分的一种肽（C5a）和白细胞介素-8（IL-8）。所有表达的受体在激动剂刺激后均经历同源磷酸化和脱敏。当共表达时，带有表位标签的C5a受体（ET-C5aR）和带有表位标签的IL-8受体（ET-IL-8RA）通过另一种受体的激活而发生交叉磷酸化。带有表位标签的fMLP受体（ET-FR）的激活也使ET-C5aR和ET-IL-8RA发生交叉磷酸化，但ET-FR对交叉磷酸化完全耐药。同样，C5a和IL-8对[35S]鸟苷5'-3-O-（硫代）三磷酸（GTPγS）结合和Ca2+动员的刺激相互之间以及与fMLP均发生交叉脱敏。然而，fMLP对[35S]GTPγS结合刺激也不会被C5a或IL-8交叉脱敏，而Ca2+动员则会，这表明在G蛋白激活远端存在一个抑制位点。与Ca2+动员的这种脱敏一致，在同时表达ET-C5aR和ET-FR的RBL-2H3细胞中，肌醇1,4,5-三磷酸释放表明fMLP和C5a相互交叉脱敏了对方刺激磷酸肌醇水解的能力。综上所述，这些结果表明受体交叉磷酸化与G蛋白激活水平的脱敏直接相关。ET-FR对此过程具有抗性。值得注意的是，在没有受体磷酸化的情况下，证明了ET-FR在磷酸肌醇水解和Ca2+动员水平上的交叉脱敏。这表明在受体/G蛋白偶联远端的一个位点存在一种新的趋化因子交叉调节形式，涉及磷脂酶C的活性。
