Regulation of human chemokine receptors CXCR4. Role of phosphorylation in desensitization and internalization.
Members of the chemokine receptor family CCR5 and CXCR4 have recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells. Here, we investigated the regulation of CXCR4 in rat basophilic leukemia cells (RBL-2H3) stably transfected with wild type (Wt CXCR4) or a cytoplasmic tail deletion mutant (DeltaCyto CXCR4) of CXCR4. The ligand, stromal cell derived factor-1 (SDF-1) stimulated higher G-protein activation, inositol phosphate generation, and a more sustained calcium elevation in cells expressing DeltaCyto CXCR4 relative to Wt CXCR4. SDF-1 and phorbol 12-myristate 13-acetate (PMA), but not a membrane permeable cAMP analog induced rapid phosphorylation as well as desensitization of Wt CXCR4. Phosphorylation of DeltaCyto CXCR4 was not detected under any of these conditions. Despite lack of receptor phosphorylation, calcium mobilization by SDF-1 in DeltaCyto CXCR4 cells was partially desensitized by prior treatment with SDF-1. Of interest, the rapid release of calcium was inhibited without affecting the sustained calcium elevation, indicating independent regulatory pathways for these processes. PMA completely inhibited phosphoinositide hydrolysis and calcium mobilization in Wt CXCR4 but only partially inhibited these responses in DeltaCyto CXCR4. cAMP also partially inhibited these responses in both Wt CXCR4 and DeltaCyto CXCR4. SDF-1, PMA, and cAMP caused phosphorylation of phospholipase Cbeta3 in Wt and DeltaCyto CXCR4 cells. Both SDF-1 as well as PMA induced rapid internalization of Wt CXCR4. SDF-1 but not PMA induced internalization of DeltaCyto CXCR4 albeit at reduced levels relative to Wt CXCR4. These results indicate that signaling and internalization of CXCR4 are regulated by receptor phosphorylation dependent and independent mechanisms. Desensitization of CXCR4 signaling, independent of receptor phosphorylation, appears to be a consequence of the phosphorylation of phospholipase Cbeta3.
Haribabu
B
B
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA. Boddu001@mc.duke.edu
Richardson
R M
RM
Fisher
I
I
Sozzani
S
S
Peiper
S C
SC
Horuk
R
R
Ali
H
H
Snyderman
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D054377:Chemokine CXCL12; D018925:Chemokines / Q000378:metabolism; D019743:Chemokines, CXC; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D054799:Phospholipase C beta; D010766:Phosphorylation; D051381:Rats; D019718:Receptors, CXCR4 / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D018925:Chemokines; D019743:Chemokines, CXC; D007527:Isoenzymes; D019718:Receptors, CXCR4; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium