Heterotrimeric G proteins couple cell surface receptors to intracellular effectors at the cytoplasmic face of membranes. The heterotrimer consists of a subunits which bind the guanine nucleotide and bg subunits which are tightly bound together. The membrane localization of G proteins is critical for their ability to relay signal between membrane-bound receptors and effectors. Palmitoylation, the reversible addition of 16 carbon palmitic acid to cysteine residues, occurs on most a subunits and participates in membrane localization. Receptor activation of as causes turnover of the palmitate group suggesting that palmitoylation regulates G protein signaling. The function of a subunit palmitoylation on bg and receptor interactions was investigated. In vitro, palmitoylated as bound bg with a 5 fold higher affinity than nonpalmitoylated as indicating that bg binding and palmitoylation can reciprocally potentiate membrane attachment of as. To evaluate receptor-a subunit coupling, membranes were treated with and without hydroxylamine to remove the palmitate and ligand binding affinity was determined. Preliminary results showed that hydroxylamine treatment caused a shift from a high affinity binding receptor state to a low affinity state suggesting that palmitoylation increased the affinity of the a subunit for the receptor. We have also investigated whether palmitoylation directs proteins to specific intracellular membranes by transfecting cells with the wild-type and palmitoylation-defective mutants of XLas. This protein is a splice variant of as with an additional 367 amino acids on the amino-terminus of as. Wild -type XLas was predominantly localized to the Golgi. A series of cysteine-to-serine mutations which gradually diminished palmitoylation of the protein did not effect distribution except to decrease membrane attachment. In contrast, deletion of a 24 amino acid proline-rich in the extra long portion reduced Golgi localization without effecting expression level or membrane attachment. A proline-rich region is also a putative signal for Golgi localization of a Gi2a splice variant suggesting that this domain could be a general signal for Golgi localization of a subunits and possibly other proteins as well.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Intramural Research (Z01)
Project #
1Z01DK043010-04
Application #
6161976
Study Section
Special Emphasis Panel (MDB)
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Bahia, Daljit S; Sartania, Nana; Ward, Richard J et al. (2003) Concerted stimulation and deactivation of pertussis toxin-sensitive G proteins by chimeric G protein-coupled receptor-regulator of G protein signaling 4 fusion proteins: analysis of the contribution of palmitoylated cysteine residues to the GAP activity o J Neurochem 85:1289-98
Osterhout, James L; Waheed, Abdul A; Hiol, Abel et al. (2003) Palmitoylation regulates regulator of G-protein signaling (RGS) 16 function. II. Palmitoylation of a cysteine residue in the RGS box is critical for RGS16 GTPase accelerating activity and regulation of Gi-coupled signalling. J Biol Chem 278:19309-16
Hiol, Abel; Davey, Penelope C; Osterhout, James L et al. (2003) Palmitoylation regulates regulators of G-protein signaling (RGS) 16 function. I. Mutation of amino-terminal cysteine residues on RGS16 prevents its targeting to lipid rafts and palmitoylation of an internal cysteine residue. J Biol Chem 278:19301-8
Waheed, Abdul A; Jones, Teresa L Z (2002) Hsp90 interactions and acylation target the G protein Galpha 12 but not Galpha 13 to lipid rafts. J Biol Chem 277:32409-12
Miura, Y; Hanada, K; Jones, T L (2001) G(s) signaling is intact after disruption of lipid rafts. Biochemistry 40:15418-23
Ugur, O; Jones, T L (2000) A proline-rich region and nearby cysteine residues target XLalphas to the Golgi complex region. Mol Biol Cell 11:1421-32
Devedjiev, Y; Dauter, Z; Kuznetsov, S R et al. (2000) Crystal structure of the human acyl protein thioesterase I from a single X-ray data set to 1.5 A. Structure 8:1137-46
Adam, L; Bouvier, M; Jones, T L (1999) Nitric oxide modulates beta(2)-adrenergic receptor palmitoylation and signaling. J Biol Chem 274:26337-43