title: Physiological requirement for both SH2 domains for phospholipase C-gamma1 function and interaction with platelet-derived growth factor receptors.
abstract: Two approaches have been utilized to investigate the role of individual SH2 domains in growth factor activation of phospholipase C-gamma1 (PLC-gamma1). Surface plasmon resonance analysis indicates that the individual N-SH2 and C-SH2 domains are able to specifically recognize a phosphotyrosine-containing peptide corresponding to Tyr 1021 of the platelet-derived growth factor (PDGF) beta receptor. To assess SH2 function in the context of the full-length PLC-gamma1 molecule as well as within the intact cell, PLC-gamma1 SH2 domain mutants, disabled by site-directed mutagenesis of the N-SH2 and/or C-SH2 domain(s), were expressed in Plcg1(-/-) fibroblasts. Under equilibrium incubation conditions (4 degrees C, 40 min), the N-SH2 domain, but not the C-SH2 domain, was sufficient to mediate significant PLC-gamma1 association with the activated PDGF receptor and PLC-gamma1 tyrosine phosphorylation. When both SH2 domains in PLC-gamma1 were disabled, the double mutant did not associate with activated PDGF receptors and was not tyrosine phosphorylated. However, no single SH2 mutant was able to mediate growth factor activation of Ca2+ mobilization or inositol 1,4,5-trisphosphate (IP3) formation. Subsequent kinetic experiments demonstrated that each single SH2 domain mutant was significantly impaired in its capacity to mediate rapid association with activated PDGF receptors and become tyrosine phosphorylated. Hence, when assayed under physiological conditions necessary to achieve a rapid biological response (Ca2+ mobilization and IP3 formation), both SH2 domains of PLC-gamma1 are essential to growth factor responsiveness.
authors.affiliation: Departments of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
authors.forename: Q S
authors.initials: QS
authors.lastname: Ji
authors.forename: A
authors.initials: A
authors.lastname: Chattopadhyay
authors.forename: M
authors.initials: M
authors.lastname: Vecchi
authors.forename: G
authors.initials: G
authors.lastname: Carpenter
mesh_terms: D000818:Animals; D002460:Cell Line; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D051966:Phospholipase C gamma; D011485:Protein Binding; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics
chemical_list: D007527:Isoenzymes; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases; D051966:Phospholipase C gamma