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Molecular and Cellular Biology, July 1999, p. 4961-4970, Vol. 19, No. 7
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Physiological Requirement for Both SH2 Domains for Phospholipase C-gamma 1 Function and Interaction with Platelet-Derived Growth Factor Receptors

Qun-sheng Ji,1 Ansuman Chattopadhyay,1 Manuela Vecchi,1,dagger and Graham Carpenter1,2,*

Departments of Biochemistry1 and Medicine,2 Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Received 25 January 1999/Returned for modification 4 March 1999/Accepted 19 April 1999

Two approaches have been utilized to investigate the role of individual SH2 domains in growth factor activation of phospholipase C-gamma 1 (PLC-gamma 1). 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-gamma 1 molecule as well as within the intact cell, PLC-gamma 1 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°C, 40 min), the N-SH2 domain, but not the C-SH2 domain, was sufficient to mediate significant PLC-gamma 1 association with the activated PDGF receptor and PLC-gamma 1 tyrosine phosphorylation. When both SH2 domains in PLC-gamma 1 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-gamma 1 are essential to growth factor responsiveness.

* Corresponding author. Mailing address: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146. Phone: (615) 322-6678. Fax: (615) 322-2931. E-mail: Graham.Carpenter{at}mcmail.vanderbilt.edu.

dagger Present address: Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy.

Molecular and Cellular Biology, July 1999, p. 4961-4970, Vol. 19, No. 7
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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