Les protéines RGS (Regulator of G-protein Signaling) sont des inhibiteurs des voies de signalisation des protéines G. RGS4 atténue l’activité de protéines G dans plusieurs tissus tel que la diminution de son activité peut accroître la sévérité de la bradycardie, cardiomyopathies liées au diabète, l’invasion de cellule cancéreuse du sein, résistance à l’insuline et intolérance au glucose. RGS4 a été localisé à la membrane plasmique ainsi que dans des compartiments intracellulaires, cependant, son mode de trafique intracellulaire reste méconnu. En utilisant des outils de microscopie confocale sur cellules vivantes et méthode de détection d’activité des voies de signalisation conditionnée par les protéines G, nous avons caractérisé l’importance de deux sites de palmitoylation, ces deux sites : Cys2 et Cys12 montrent des intérêts complémentaires dans le trafic de RGS4 vers la membrane cellulaire. Dans un axe linéaire, nous avons identifié DHHC3 et 7, deux enzymes de palmitoylation participant au trafique intracellulaire de RGS4 et donc à la maximalisation de son activité inhibitrice des voies de signalisation contrôlées par Galphaq. Enfin des marqueurs de membranes endosomales, les protéines rab ont permis de caractériser les voies de trafic intracellulaire empruntée par RGS4, par exemple RGS4 est internalisé de la membrane plasmique par Rab5 et recyclé à la membrane cellulaire par Rab11. L’activation ou inhibition de Rab5 et 11 ont permis d’observer des changements d’activité de RGS4. Ces travaux confèrent une base de données pour des études ultérieures visant à développer des stratégies thérapeutiques à accroître les fonctionnalités de RGS4. / RGS proteins (Regulator of G-protein Signaling) are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues such that loss of its function may lead to bradycardia, diabetic cardiomyopathy, breast cancer cell invasion, insulin resistance and glucose intolerance. RGS4 has been localized to both plasma membrane and intracellular pools, however, the nature of its intracellular trafficking remains to be elucidated. G-protein inhibition requires the presence of RGS4 at the plasma membrane. In this work, we characterized the complementary roles of two putative palmitoylation sites on RGS4 to target intracellular compartments and plasma membrane. We identified palmitoylation on Cys2 and 12 respectively important for RGS4 endosomal targeting and plasma membrane localization, when mutations were introduced to the palmitoylation sites, RGS4 capability of inhibiting Gq-mediated signaling was impaired. As a continuum we identified two palmitoylating enzymes, DHHC3 and 7 as modulator of RGS4 localization and function. Knock downs of DHHC3 and 7 impaired RGS4 endosomal and plasma membrane targeting and capability of inhibiting M1-muscarinic receptor signaling. Finally we used live cell confocal microscopy to define RGS4 intracellular trafficking routes. Specifically Rab5 mediated RGS4 trafficking from the plasma membrane to intracellular compartments while Rab11 mediated RGS4 trafficking to the plasma membrane. Activation and inhibition of Rab5 and 11 routes impaired RGS4 capability of inhibiting M1-muscarinic receptor signaling pathway. These novel findings provide a strong rationale for future studies aimed at developing new strategies to increase the function of RGS4.

Regulator of G-protein signalling 4 (RGS4) modulates Gq and Gi signalling at the plasma membrane (PM). It has been demonstrated that the addition of palmitate to cysteine residues is an important regulator of RGS protein localization and function. The family of palmitate transferase enzymes shares a conserved Asp-His-His-Cys (DHHC) motif. We set out to establish the DHHC isoform(s) that affect RGS4 activity in HEK201 cells. Confocal microscopy revealed that overexpression of DHHCs 3 and 7 mobilized RGS4 to the Golgi. Knockdown of either DHHC3 or DHHC7 attenuated RGS4 inhibition of Gαq-coupled Ca2+ release and reduced RGS4 PM localization. Consistent with a role in promoting RGS4 lipid bilayer targeting, dominant negative mutants of the five most highly expressed DHHCs in HEK201 cells also diminished RGS4 PM association. Together, these data suggest that members of the mammalian DHHC family regulate RGS4 localization and function, likely through palmitoylation of its target cysteine residues.

Regulator of G-protein signalling 4 (RGS4) modulates Gq and Gi signalling at the plasma membrane (PM). It has been demonstrated that the addition of palmitate to cysteine residues is an important regulator of RGS protein localization and function. The family of palmitate transferase enzymes shares a conserved Asp-His-His-Cys (DHHC) motif. We set out to establish the DHHC isoform(s) that affect RGS4 activity in HEK201 cells. Confocal microscopy revealed that overexpression of DHHCs 3 and 7 mobilized RGS4 to the Golgi. Knockdown of either DHHC3 or DHHC7 attenuated RGS4 inhibition of Gαq-coupled Ca2+ release and reduced RGS4 PM localization. Consistent with a role in promoting RGS4 lipid bilayer targeting, dominant negative mutants of the five most highly expressed DHHCs in HEK201 cells also diminished RGS4 PM association. Together, these data suggest that members of the mammalian DHHC family regulate RGS4 localization and function, likely through palmitoylation of its target cysteine residues.

Exocytosis, the fusion of a vesicle to a cellular membrane, involves a protein named SNAP-25. This protein, containing two alpha helices connected with a linker region, is localized to the cell membrane via palmitic acids attached to the cysteine residues of its linker region in a process called palmitoylation. Are cysteine residues of the SNAP-25 linker region palmitoylated in an ordered manner and to a particular extent? The answer to this question may give insight into the regulated nature of exocytosis. While it is generally accepted that SNAP-25 must be palmitoylated in order to perform its exocytotic functions, the details surrounding this process are still being discovered, defined, and understood. In these studies we replicate the oxidation, reduction, and palmitoylation of SNAP-25 in vitro. Palmitoylating SNAP-25 in vitro, a process which occurs regularly in vivo, allows us to determine the extent of palmitoylation. In vitro palmitoylation of SNAP-25 was studied both with and without a native palmitoyl acyl transferase (PAT), DHHC-17, the enzyme to attach palmitic acids to cysteines in the linker region of SNAP-25. These studies were done under a variety of conditions designed to identify (1) components necessary for optimal palmitoylation and (2) extent of palmitoylation with components that mimic native conditions. Palmitoylation is a common modification for a variety of proteins, both soluble and membrane-bound. Like phosphorylation, palmitoylation is reversible and may play an important role in regulation of cellular processes. Specifically, the palmitoylation of SNAP-25 may play a critical role in the regulation of exocytosis and therefore learning further details about this important process may help us to better understand a variety of neurodegenerative diseases and states of decreased or compromised exocytosis.