TRYPTOPHAN-650 OF HUMAN GRANULOCYTE-COLONY-STIMULATING FACTOR (G-CSF)RECEPTOR, IMPLICATED IN THE ACTIVATION OF JAK2, IS ALSO REQUIRED FOR G-CSF - MEDIATED ACTIVATION OF SIGNALING COMPLEXES OF THE P21RAS ROUTE

Granulocyte colony-stimulating factor (G-CSF) induces rapid phosphoryl ation of JAK kinases as well as activation of the p21ras route through interaction with its specific receptor (G-CSF-R). The cytoplasmic mem brane-proximal region of G-CSF-R (amino acids 631 to 684) is necessary for proliferation induction and activation of JAK2. In contrast, acti vation of She and Syp, signaling molecules implicated in the p21ras si gnaling route, depends on the phosphorylation of tyrosine residues loc ated in the membrane-distal region (amino acids 685 to 813) of G-CSF-R , We investigated whether G-CSF-induced activation of signaling comple xes of the p21ras route depends on the function of the membrane-proxim al cytoplasmic region of G-CSF-R. A G-CSF-R mutant was constructed in which tryptophan 650 was replaced by arginine and expressed in BAF3 ce lls (BAF/W650R). In contrast to BAF3 cell transfectants expressing wil d-type G-CSF-R, BAF/W650R cells did not proliferate and did not show a ctivation of JAK2, STAT1, or STAT3 in response to G-CSF. Immunoprecipi tations with anti-She and anti-Grb2 antisera showed that mutant W650R also failed to activate Syp and She. These data indicate that the memb rane-proximal cytoplasmic domain of G-CSF-R is not only crucial for pr oliferative signaling and activation of JAK2 and STATs, but is also re quired for activation of the p21ras route, which occurs via the membra ne-distal region of G-CSF-R. (C) 1996 by The American Society of Hemat ology.