Phospholipase C-G protein-coupled receptor kinase (GRK) chimeras reveal that the PH domain is a modular regulatory structure

J. W. Lomasney*, J. Ding, K. King

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

The pleckstrin homology (PH) domain of phospholipase C delta l (PLC81) has been shown to regulate enzyme activity by both serving as an anchor and as an allosteric regulatory domain by binding phosphoinositides (IP). This study tests the hypotheses that the site of regulation of GRK2 by IP also resides in its PH domain, and that the PH domain of GRK2 when fused to the catalytic center of PLCSl can regulate hydrolysis. Purified native PLC81, GRK2-PLC51 chimera, and PH domain truncation mutant DEL70-PLC51 were characterized using a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) dodecyl maltoside mixed micelle assay. Initial rates of hydrolysis of PI(4,5)P2 were similar for both native PLCSl (Vmax=39.7 umol/mg/min) and the GRK2-PLCS1 (33.4), and greater than the rates for DEL70-PLC81 (7.3) when assessed as a function of increasing bulk concentration and mol fraction (CASE ffl conditions). Curves for all three enzymes were sigmoidal demonstrating cooperativity with Hill coefficients (HILLCO) ranging from 2.3 to 2.7. Assays were also conducted under CASE I conditions in which the bulk concentration of substrate is varied while the mol fraction is fixed. Native PLCSl and the GRK2-PLC81 had greater affinity for micellar substrate compared to DEL70-PLC81; interfacial binding constant Ks=8.2uM, 8.6, and 18.9 respectively. Additionally, PLC81 and GRK2PLC81 had activities greater than DEL70-PLC81; Vmax=15.9 +/- 0.26 umol/mg/min, 13.6 +/- 0.34, and 2.7 +/- 0.1 respectively. HILLCO varied from 0.91 to 0.75. These studies suggest that the PH domain of GRK2 binds PI(4,5)P; and that the PH domain is a modular regulatory structure.

Original languageEnglish (US)
Pages (from-to)A1381
JournalFASEB Journal
Volume12
Issue number8
StatePublished - 1998

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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