Covalent lipid modifications anchor numerous signaling proteins, including heterotrimeric G proteins, nonreceptor tyrosine kinases and Ras to the cytoplasmic face of the plasma membrane where they convey signals from cell surface receptors to intracellular effectors. The posttranslational attachment of lipids to these signal transducers is necessary for their function. Palmitoylation (S-acylation) is the only covalent lipid modification that is reversible and is a regulated modification. The broad goal of this project is to understand the biology and enzymology of protein palmitoylation in signal transduction using biochemical and genetic approaches. Heterotrimeric G proteins have been used as a model to understand the functional significance of palmitoylation. In the previous grant period, a protein palmitoytransferase activity that acylates G-protein alpha subunits and Ras was identified and characterized. In this application, purification of this enzyme and cloning of its cDNA is proposed. A genetic analysis of G-protein palmitoylation in yeast revealed that both alpha and gamma subunits are modified with palmitate and provided evidence for the importance of palmitoylation in G-protein function. These studies established the basis for the genetic screens proposed in this application to identify genes that are required for palmitoylation of G-protein subunits, a necessary first step in characterizing the physiological role of G-protein palmitoyltransferases. To increase our understanding of the organization of signaling proteins in cellular membranes, the hypothesis that palmitoylation of G proteins is responsible for their targeting to specialized regions of the plasma membrane will be explored. The association of modified and unmodified G proteins with membranes enriched in glycolipids and cholesterol will be studied in artificial membranes and in cells. The recently discovered family of proteins called Regulators of G protein Signaling, or RGS proteins, revealed a novel mode of regulating signal transduction. We determined that RGS4 is palmitoylated in a domain of the protein that is required for its plasma membrane localization. The studies proposed in this application will investigate the mechanism of membrane association of RGS4 and the function and regulation of RGS4 palmitoylation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051466-07
Application #
6180566
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Cole, Alison E
Project Start
1994-08-01
Project End
2002-07-31
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
7
Fiscal Year
2000
Total Cost
$286,135
Indirect Cost
Name
Washington University
Department
Physiology
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Linder, Maurine E; Jennings, Benjamin C (2013) Mechanism and function of DHHC S-acyltransferases. Biochem Soc Trans 41:29-34
Nishimura, Akiyuki; Linder, Maurine E (2013) Identification of a novel prenyl and palmitoyl modification at the CaaX motif of Cdc42 that regulates RhoGDI binding. Mol Cell Biol 33:1417-29
Hilgemann, Donald W; Fine, Michael; Linder, Maurine E et al. (2013) Massive endocytosis triggered by surface membrane palmitoylation under mitochondrial control in BHK fibroblasts. Elife 2:e01293
Lai, Jianbin; Linder, Maurine E (2013) Oligomerization of DHHC protein S-acyltransferases. J Biol Chem 288:22862-70
Jennings, Benjamin C; Linder, Maurine E (2012) DHHC protein S-acyltransferases use similar ping-pong kinetic mechanisms but display different acyl-CoA specificities. J Biol Chem 287:7236-45
Jia, Lixia; Linder, Maurine E; Blumer, Kendall J (2011) Gi/o signaling and the palmitoyltransferase DHHC2 regulate palmitate cycling and shuttling of RGS7 family-binding protein. J Biol Chem 286:13695-703
Ahearn, Ian M; Tsai, Frederick D; Court, Helen et al. (2011) FKBP12 binds to acylated H-ras and promotes depalmitoylation. Mol Cell 41:173-85
Hang, Howard C; Linder, Maurine E (2011) Exploring protein lipidation with chemical biology. Chem Rev 111:6341-58
Aittaleb, Mohamed; Nishimura, Akiyuki; Linder, Maurine E et al. (2011) Plasma membrane association of p63 Rho guanine nucleotide exchange factor (p63RhoGEF) is mediated by palmitoylation and is required for basal activity in cells. J Biol Chem 286:34448-56
Jernigan, Kristin K; Cselenyi, Christopher S; Thorne, Curtis A et al. (2010) Gbetagamma activates GSK3 to promote LRP6-mediated beta-catenin transcriptional activity. Sci Signal 3:ra37

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