Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2.
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.
Richardson
R M
RM
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA. richa021@mc.duke.edu
Pridgen
B C
BC
Haribabu
B
B
Snyderman
R
R
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D002460:Cell Line; D004705:Endocytosis; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D054799:Phospholipase C beta; D010766:Phosphorylation; D051381:Rats; D020239:Receptor Cross-Talk; D054389:Receptors, CCR1; D019707:Receptors, Chemokine / Q000378:metabolism*; D018123:Receptors, Interleukin / Q000378:metabolism*; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D010738:Type C Phospholipases / Q000378:metabolism
D015703:Antigens, CD; C515300:CCR1 protein, human; C515302:Ccr1 protein, rat; D016209:Interleukin-8; D007527:Isoenzymes; D054389:Receptors, CCR1; D019707:Receptors, Chemokine; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D010738:Type C Phospholipases; D054799:Phospholipase C beta