Interaction with Gβγ Is Required for Membrane Targeting and Palmitoylation of Gαs and Gαq

Abstract

Peripheral membrane proteins utilize a variety of mechanisms to attach tightly, and often reversibly, to cellular membranes. The covalent lipid modifications, myristoylation and palmitoylation, are critical for plasma membrane localization of heterotrimeric G protein alpha subunits. For alpha(s) and alpha(q), two subunits that are palmitoylated but not myristoylated, we examined the importance of interacting with the G protein betagamma dimer for their proper plasma membrane localization and palmitoylation. Conserved alpha subunit N-terminal amino acids predicted to mediate binding to betagamma were mutated to create a series of betagamma binding region mutants expressed in HEK293 cells. These alpha(s) and alpha(q) mutants were found in soluble rather than particulate fractions, and they no longer localized to plasma membranes as demonstrated by immunofluorescence microscopy. The mutations also inhibited incorporation of radiolabeled palmitate into the proteins and abrogated their signaling ability. Additional alpha(q) mutants, which contain these mutations but are modified by both myristate and palmitate, retained their localization to plasma membranes and ability to undergo palmitoylation. These findings identify binding to betagamma as a critical membrane attachment signal for alpha(s) and alpha(q) and as a prerequisite for their palmitoylation, while myristoylation can restore membrane localization and palmitoylation of betagamma binding-deficient alpha(q) subunits.

Heterotrimer Formation, Together with Isoprenylation, Is Required for Plasma Membrane Targeting of Gऔγ

“…Constructs-Wild type ␣ s (HA-tagged) and ␤-binding-deficient mutant ␣ s IEKϩ(HA-tagged) were described previously (10). The expression vector for ␤ 1 (Myc-and His-tagged) was provided by David P. Siderovski (University of North Carolina) (9).…”

“…Cell Fractionation Assay-Soluble and particulate fractions were isolated as described previously (10). Briefly, 48 h after transfection HEK293 cells were washed in ice-cold PBS and lysed in hypotonic lysis buffer (50 mM Tris-HCl, pH 8, 2.5 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol) with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, 5 g/ml aprotinin).…”

“…Expression of ␤␥ recovers palmitoylation and PM localization of non-myristoylated G2A mutants of ␣ i and ␣ z . Furthermore, inhibiting ␣ and ␤␥ interaction by expression of a ␤␥-sequestering protein (30) or by mutating ␤␥ contact sites in ␣ subunits decreases palmitoylation and PM localization of ␣ z (11) or ␣ s (10). Targeting of ␤ 1 ␥ 2 to mitochondria through a mito-␥ 2 mutant also directs co-expressed ␣ z to mitochondria (11).…”

Functional consequences of Gα13 mutations that disrupt interaction with p115RhoGEF

“…A control experiment showed that a q was effectively pulled down by b 1 g 2 , whereas a mutant of a q (a q IE), previously reported to be b 1 g 2 -binding defective, did not show any binding (Figure 7) (Evanko et al, 2000). The point mutants a 13 G205S, a 13 K204E, a 13 E229K, a 13 R232E, and the double mutants a 13 K204E/E229K and a 13 E229K/ R232E also bound to Myc-His b 1 g 2 dimers, indicating that these mutations do not affect the structural integrity of a 13 .…”

Light-regulated translocation of signaling proteins in Drosophila photoreceptors

“…All α subunits of heterotrimeric G-proteins (with the exception of transducin) are reversibly modified by palmitoylation, i.e., the attachment of palmitate to a cysteine residue near the N-terminus (reviewed in Chen and Manning (2001), Mumby (1997), Smotrys and Linder (2004), Wedegaertner (1998)). The second major determinant of G-protein localization is the anchoring of the Gβγ subunits to the membrane (Evanko et al, 2001(Evanko et al, ,2000.…”