Abstract
Pleckstrin homology (PH) domains are 90-110 amino acid regions of protein sequence homology that are found in a variety of proteins involved in signal transduction and growth control. We have previously reported that the PH domains of several proteins, including beta ARK1, PLC gamma, IRS-1, Ras-GRF, and Ras-GAP, expressed as glutathione S-transferase fusion proteins, can reversibly bind purified bovine brain G beta gamma subunits in vitro with varying affinity. To determine whether PH domain peptides would behave as antagonists of G beta gamma subunit-mediated signal transduction in intact cells, plasmid minigene constructs encoding these PH domains were prepared, which permit transient cellular expression of the peptides. Pertussis toxin-sensitive, G beta gamma subunit-mediated inositol phosphate (IP) production was significantly inhibited in COS-7 cells transiently coexpressing the alpha 2-C10 adrenergic receptor (AR) and each of the PH domain peptides. Pertussis toxin-insensitive, Gq alpha subunit-mediated IP production via coexpressed M1 muscarinic acetylcholine receptor (M1 AChR) was attenuated only by the PLC gamma PH domain peptide, suggesting that the inhibitory effect of most of the PH domain peptides was G beta gamma subunit-specific. Stimulation of the mitogen-activated protein (MAP) kinase pathway by Gi-coupled receptors in COS-7 cells has been reported to require activation of p21ras and to be independent of protein kinase C. Since several proteins involved in activation contain PH domains, the effect of PH domain peptide expression on alpha 2-C10 AR-mediated p21ras-GTP exchange and MAP kinase activation as well as direct G beta gamma subunit-mediated activation of MAP kinase was determined. In each assay, coexpression of the PH domain peptides resulted in significant inhibition. Increasing G beta gamma subunit expression surmounted PH domain peptide-mediated inhibition of MAP kinase activation. These data suggest that the PH domain peptides behave as specific antagonists of G beta gamma-mediated signaling in intact cells and that interactions between PH domains and G beta gamma subunits or structurally related proteins may play a role in the activation of mitogenic signaling pathways by G protein-coupled receptors.
Highlights
From the Howard Hughes Medical Institute and the Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Included in the list of approximately 70 known Pleckstrin homology (PH) domain-containing proteins are several that participate in the function ofRas including Ras-GRF, Sosl, and Ras-GAP, the growth factor-binding protein Grb7, the insulin receptor substrate IRS-I, serine/threonine kinases including Rae-a, Rae-B. and the t3-adrenergic receptor kinases t3ARKI and t3ARK2, tyrosine kinases including the Bruton tyrosine kinase (Btk), Tee, and TSK, phospholipase C species including PLC'Y and PLCa, the cytoskeletal protein spectrin, the microtubule-binding GTPase dynamin, and oxysterol binding protein [3,4,5]
We have previously shown that the Gf3'Ybinding I3ARKI PH domain [10,11,12] antagonizes a2-CI0 AR-mediated inositol phosphate (IP) production with no effect on that mediated by the muscarinic acetylcholine receptor (Ml AChR) [24] in COS-7 cells
Summary
Materials-The cDNA for the human a2-CI0 AR was cloned in our laboratory [17]. The cDNA for the human Ml AChR [18] was provided by Dr Ernest Peralta. Transfected COS-7 cells in 6-well plates, preincubated overnight in low serum medium (DMEM, 0.5% fetal bovine serum), were stimulated for 5 min with the indicated agonist, washed with ice-cold calcium- and magnesium-free phosphate-buffered-saline, lysed in 200 /Ll of ice-cold lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 10 mM sodium pyrophosphate, 0.1 mM phenylmethylsulfonyl fluoride) and clarified by centrifugation. Activation of p21 ra'-Transfected COS-7 cells in 6-well plates were serum-starved overnight as described, labeled for 2 h in phosphate-free DMEM containing [32Plorthophosphate (200 /LCi/ml), and stimulated for 2 min with or without UK-14304. Labeled GDP and GTP were quantitated using a Molecular Dynamics PhosphorImager, and data were expressed as the percentage of GTP over total labeled guanine nucleotides
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