Richardson R M, Pridgen B C, Haribabu B, Snyderman R
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2000 Mar 31;275(13):9201-8. doi: 10.1074/jbc.275.13.9201.
To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1alpha, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca(2+) mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca(2+) mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (DeltaCCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca(2+) mobilization. S2, S3, and DeltaCCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co-expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca(2+) mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca(2+) mobilization by S3 and DeltaCCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and DeltaCCR1, which produced sustained signals, cross-phosphorylated and cross-desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization) which is inversely correlated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation.
为了研究CCR1趋化因子受体的调节机制，对大鼠嗜碱性白血病（RBL-2H3）细胞系进行改造，使其稳定表达表位标记的受体。这些细胞对RANTES（活化时正常T细胞表达和分泌的调节因子）、巨噬细胞炎性蛋白-1α和单核细胞趋化蛋白-2产生反应，介导磷脂酶C激活、细胞内Ca(2+)动员和胞吐作用。激活后，通过GTPase刺激减弱和Ca(2+)动员来测定，CCR1发生磷酸化和脱敏。CCR1特定丝氨酸和苏氨酸残基（S2和S3）的丙氨酸替代或CCR1胞质尾的截断（DeltaCCR1）消除了受体磷酸化和G蛋白激活的脱敏，但未消除Ca(2+)动员的脱敏。相对于S1和CCR1，S2、S3和DeltaCCR1也对内化有抗性，介导更大的磷脂酰肌醇水解和持续的Ca(2+)动员，并且仅被RANTES部分脱敏。为了研究CCR1的交叉调节，制备了共表达CCR1和白细胞介素-8受体（CXCR1、CXCR2或CXCR2的磷酸化缺陷突变体331T）的RBL细胞。白细胞介素-8对CXCR1或CXCR2的刺激使CCR1发生交叉磷酸化，并使其刺激GTPase活性和Ca(2+)动员的能力交叉脱敏。有趣的是，CCR1使CXCR2发生交叉磷酸化和交叉脱敏，但对CXCR1没有作用。尽管缺乏受体磷酸化，S3和DeltaCCR1的Ca(2+)动员也被CXCR1和CXCR2交叉脱敏。与野生型CCR1相反，产生持续信号的S3和DeltaCCR1对CXCR1以及CXCR2的反应进行交叉磷酸化和交叉脱敏。综上所述，这些结果表明CCR1介导的反应在信号通路的几个步骤中受到调节，在受体/G蛋白偶联水平通过受体磷酸化，在磷脂酶C激活水平通过未知机制。此外，趋化因子受体之间的选择性交叉调节部分是信号强度的结果（即更大的磷脂酰肌醇水解和持续的Ca(2+)动员），这与受体对磷酸化的敏感性呈负相关。由于许多趋化因子激活多种趋化因子受体，此类受体之间的选择性交叉调节可能在其免疫调节中起作用。
