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Defining the mechanisms by which palmitoylation regulates the localization and function of RGS4
Author(s) -
Dissanayake Kaveesh,
Bastin Guillaume,
Heximer Scott P
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.904.1
Subject(s) - palmitoylation , hek 293 cells , heterotrimeric g protein , microbiology and biotechnology , transfection , g protein , biology , chemistry , signal transduction , biochemistry , cysteine , receptor , gene , enzyme
Regulators of G‐Protein Signalling (RGS) proteins are strong candidates for modulating cardiovascular signalling based on their high expression in cardiac and sinoatrial node myocytes, and their ability to potently inhibit heterotrimeric G‐proteins. RGS4 regulates G‐protein function at the plasma membrane (PM) and it has been demonstrated that palmitoylation, the post‐translational addition of a 16 carbon palmitate group to cysteine, is important for RGS4 translocation to the PM. Confocal microscopy data demonstrated that mutation of cysteine residues 2 and 12 removed RGS4 localization to the PM. Calcium imaging confirmed that RGS4‐C2A,C12A elicited less inhibition of Gq‐dependent calcium release than wildtype in HEK293T cells. A library of 23 mammalian palmitoyl‐acyl transferase enzymes has been cloned and isolated, each sharing a conserved catalytic Asp‐His‐His‐Cys (DHHC) site [Fukata et al, 2006]. We set out to establish the DHHC isoforms that regulate the localization and function of RGS4 in HEK cells. We created dominant negative mutations of five DHHCs most highly expressed in HEK cells. Confocal microscopy data showed that co‐transfection of mutant DHHCs 3 and 7 with RGS4 removed localization of RGS4 to the PM. Further studies will examine changes in Gq‐dependent calcium release following transfection of RGS4 in HEK cells where target DHHCs are knocked down using siRNAs. This study was funded by the Heart and Stroke Foundation of Ontario Grant‐in‐Aid Program grants T6799 and the Heart and Stroke/Richard Lewar Centre of Excellence Studentship Award.