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.

Mutation of G␣ q or G␣ s N-terminal contact sites for G␤␥ resulted in ␣ subunits that failed to localize at the plasma membrane or undergo palmitoylation when expressed in HEK293 cells. We now show that overexpression of specific ␤␥ subunits can recover plasma membrane localization and palmitoylation of the ␤␥-bindingdeficient mutants of ␣ s or ␣ q . Thus, the ␤␥-bindingdefective ␣ is completely dependent on co-expression of exogenous ␤␥ for proper membrane localization. In this report, we examined the ability of ␤ 1-5 in combination with ␥ 2 or ␥ 3 to promote proper localization and palmitoylation of mutant ␣ s or ␣ q . Immunofluorescence localization, cellular fractionation, and palmitate labeling revealed distinct subtype-specific differences in ␤␥ interactions with ␣ subunits. These studies demonstrate that 1) ␣ and ␤␥ reciprocally promote the plasma membrane targeting of the other subunit; 2) ␤ 5 , when coexpressed with ␥ 2 or ␥ 3 , fails to localize to the plasma membrane or promote plasma membrane localization of mutant ␣ s or ␣ q ; 3) ␤ 3 is deficient in promoting plasma membrane localization of mutant ␣ s and ␣ q , whereas ␤ 4 is deficient in promoting plasma membrane localization of mutant ␣ q ; 4) both palmitoylation and interactions with ␤␥ are required for plasma membrane localization of ␣. G proteins1 are found on the cytoplasmic face of the plasma membrane (PM) where they transduce signals from heptahelical receptors to effector proteins (1, 2). Activation of the receptor via agonist binding induces a conformational change in the G protein ␣␤␥ heterotrimer, which triggers its dissociation from the receptor in the form of a GTP-bound ␣ subunit and a ␤␥ complex. Each subunit is then capable of regulating the function of various effector proteins. Because proper membrane localization is a prerequisite for the correct functioning of this system, numerous studies have examined the requirements for targeting of the G protein ␣ subunits to the plasma membrane. The two major requirements seem to be covalent lipid modifications (3, 4) and binding to the ␤␥ complex (5, 6).Two types of lipid modification of G protein ␣ subunits have been described, both of which occur at the extreme N terminus of the protein. These subunits are either palmitoylated, in the case of ␣ s , ␣ q , ␣ 12 , and ␣ 13 , or myristoylated and palmitoylated in the case of ␣ i , ␣ o , and ␣ z . Myristate, a 14-carbon fatty acid, is attached co-translationally and irreversibly, whereas palmitate, a 16-carbon fatty acid, is attached post-translationally and reversibly. These lipid modifications help anchor the G protein ␣ subunit to the PM, but in the case of ␣ s and ␣ q , and presumably ␣ 12 and ␣ 13 as well, palmitoylation and proper localization of the ␣ subunit at the PM requires stable binding between the ␣ and ␤␥ subunits (6). The ␤␥ complex is anchored at the PM with the help of its own lipid modification, the post-translational attachment of either a farnesyl or geranylgeranyl moiety to a cysteine at a C-terminal CAAX box on the ␥ s...

G protein-coupled receptor kinases (GRKs) specifically phosphorylate agonist-occupied G protein-coupled receptors at the inner surface of the plasma membrane (PM), leading to receptor desensitization. GRKs utilize a variety of mechanisms to bind tightly, and sometimes reversibly, to cellular membranes. Previous studies demonstrated the presence of a membrane binding domain in the C terminus of GRK5. Here we define a mechanism by which this short C-terminal stretch of amino acids of GRK5 mediates PM localization. Secondary structure predictions suggest that a region contained within amino acids 546 -565 of GRK5 forms an amphipathic helix, with the key features of the predicted helix being a hydrophobic patch of amino acids on one face of the helix, hydrophilic amino acids on the opposite face, and a number of basic amino acids surrounding the hydrophobic patch. We show that amino acids 546 -565 of GRK5 are sufficient to target the cytoplasmic green fluorescent protein (GFP) to the PM, and the hydrophobic amino acids are necessary for PM targeting of GFP-546 -565. Moreover, full-length GRK5-GFP is localized to the PM, but mutation of the hydrophobic patch or the surrounding basic amino acids prevents PM localization of GRK5-GFP. Last, we show that mutation of the hydrophobic residues severely diminishes phospholipid-dependent autophosphorylation of GRK5 and phosphorylation of membrane-bound rhodopsin by GRK5. The findings in this report thus suggest the presence of a membrane binding motif in GRK5 and define the importance of a group of hydrophobic amino acids within this motif in mediating its PM localization.Cell surface localized G protein-coupled receptors (GPCRs) 1 detect a large variety of extracellular stimuli and initiate numerous intracellular signaling pathways. Proper regulation of GPCR signaling is maintained in part by a process known as desensitization (1), which refers to the turning off of a GPCRinitiated signaling event in the continuous presence of agonist. A key mechanism of GPCR desensitization is phosphorylation of agonist-occupied receptor by the G protein-coupled receptor kinases (GRKs) (2, 3). This phosphorylation promotes binding of arrestin proteins to the GPCR and results in subsequent uncoupling of the GPCR from G protein.To function properly GRKs need to be localized to the cytoplasmic face of the plasma membrane (PM) where their GPCR substrates are located. Different members of the GRK family utilize a variety of mechanisms to bind to cellular membranes (2, 3). GRK2 and GRK3 contain well characterized C-terminal pleckstrin homology domains (4) that mediate translocation of the kinases from the cytoplasm to the PM in response to GPCR activation (5, 6). The pleckstrin homology domains allow PM binding by interacting with both phospholipids and free G protein ␤␥ subunits. In contrast to GRK2/3, other GRK family members exhibit a more constitutive association with cellular membranes. GRK1 and GRK7 contain a C-terminal CAAX motif, which directs covalent modification by a farnesyl or gerany...

It is becoming evident that glia, and astrocytes in particular, are intimately involved in neuronal signaling. Astrocytic modulation of signaling in neurons appears to be mediated by the release of neuroactive compounds such as the excitatory amino acid glutamate. Release of these transmitters appears to be driven by two different processes: (1) a volume regulatory response triggered by hypo-osmotic conditions that leads to the release of osmotically active solutes from the cytoplasm into the extracellular space, and (2) intracellular calcium-dependent vesicle-mediated excytotic release. The regulatory volume decrease may be mediated by any of several different pathways that increase membrane permeability, thus allowing osmolytes to travel down their concentration gradient into the extracellular space. Such pathways include anion channels, hemichannels, P2X receptor channels, and transporters or multidrug resistance proteins. The excytotic release process may use calcium triggered synaptic like vesicle fusion or alterations in constitutive vesicle trafficking to the membrane. Determining the contribution of any of these release mechanisms requires agents that can be used to specifically block pathways of interest. Currently, many of the pharmacological compounds being used exhibit a great deal of cross-reactivity between several of these pathways. For example, the popular anion channel inhibitor 5-nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB) is an efficient blocker of both hemichannels and vesicle loading. This demonstrates the need to more fully characterize the activities of the agents currently available and to choose pathway blockers carefully when designing experiments.

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