Heterotrimeric G protein a,b, and g subunits are subject to several kinds of co-and post-translational covalent modi®cations. Among those relevant to G proteincoupled receptor signaling in normal cell function are lipid modi®cations and phosphorylation. N-myristoylation is a co-translational modi®cation occurring for members of the G i family of Ga subunits, while palmitoylation is a post-translational modi®cation that occurs for these and most other Ga subunits. One or both modi®cations are required for plasma membrane targeting and contribute to regulating strength of interaction with the Gbg heterodimer, eectors, and regulators of G protein signaling (RGS proteins). Ga subunits, including those with transforming activity, are often inactive when unable to be modi®ed with lipids. The reversible nature of palmitoylation is intriguing in this regard, as it lends itself to a regulation integrated with the activation state of the G protein. Several Ga subunits are substrates for phosphorylation by protein kinase C and at least one is a substrate for phosphorylation by the p21-activated protein kinase. Phosphorylation in both instances inhibits the interactions of these subunits with the Gbg heterodimer and RGS proteins. Several Ga subunits are also substrates for tyrosine phosphorylation. A Gg subunit is phosphorylated by protein kinase C, with the consequence that it interacts more tightly with a Ga subunit but less well with an eector. Oncogene (2001) 20, 1643 ± 1652.

[11C]WIN 35,428 was evaluated as a specific in vivo radioligand for the dopamine transporter site by PET scanning in nonhuman primates and humans. In studies with a baboon (Papio anubis), [11C]WIN 35,428 accumulated in brain regions containing dopamine transporters, i.e., the striata. This accumulation was partially blocked by prior administration of (-)cocaine (4 mg/kg, i.v.). Placement of a unilateral lesion of dopamine-containing nerve terminals with MPTP resulted in a unilateral reduction in [11C]WIN 35,428 accumulation in the striatum on the side of the lesion. Imaging of D2 dopamine receptors with [11C]NMSP in the same MPTP-treated animals showed much less reduction in the postsynaptic D2 dopamine receptors as compared to the much larger reduction in the dopamine transporters labeled with [11C]WIN 35,428. A total of ten normal human volunteers (five males and five females) with ages ranging from 19 to 81 years were studied. The caudate/cerebellar and putamen/cerebellar ratios ranged from 4.4 to 5.7 90 min after injection of the tracer. Preliminary kinetic modeling with arterial plasma sampling resulted in an average binding potential (k3/k4) of 4.98 in the caudate nucleus and 5.13 in putamen. To demonstrate in vivo blockade with dopamine reuptake inhibitors, two subjects received prior oral doses of 6 mg mazindol. Subject 5 had significant reductions of 29% in the caudate/cerebellar ratio at 90 min, 35% in the putamen/cerebellar ratio at 90 min, 45% in the caudate k3/k4 ratio from 6.7 to 3.7, and 46% in the putamen k3/k4 from 4.7 to 2.5. Subject 8 had significant reductions of 20% in both the caudate/cerebellar ratio and the putamen/cerebellar ratio at 90 min. During the human PET studies, a number of neuropsychological tests and physiological measurements were performed. No significant changes were found after administration of the [11C]WIN 35,428 alone. Taken together, these data indicate that [11C]WIN 35,428 is a promising radioligand for future studies of neuropsychiatric disorders that involve the dopamine transporter site.

Many of the ␣ subunits of heterotrimeric GTP-binding regulatory proteins (G proteins) are palmitoylated, a modification proposed to play a key role in the stable anchorage of the subunits to the plasma membrane. Palmitoylation of ␣ subunits from the G i family is preceded by N-myristoylation, which alone or together with ␤␥ probably supports a reversible interaction of the ␣ subunit with membrane as a prerequisite to the eventual incorporation of palmitate. Previous studies have not addressed, however, the question of whether membrane association alone, carried out through N-myristoylation, interaction with ␤␥, or other events, is sufficient for palmitoylation. We report here for ␣ o that it is not. We found that N-myristoylation is required for palmitoylation at least in part because it supports events subsequent to membrane attachment. Mutants of ␣ o designed to target the subunit to membrane without an N-myristoyl group are unable to be palmitoylated as evaluated by incorporation of [ 3 H]palmitate. Mutants of ␣ o unable to interact normally with ␤␥ yet still attach to membrane demonstrate that ␤␥, in contrast, is not required for palmitoylation. ␤␥ becomes necessary, however, when the N-myristoyl group is absent. Our results suggest that N-myristoylation and ␤␥, while almost certainly relevant to the reversible interaction of ␣ o with membrane, also play at least partly overlapping, postanchorage roles in palmitoylation.Fatty acid acylation is one of the major covalent modifications of heterotrimeric GTP-binding regulatory protein (G protein) ␣ subunits (1-4). For most ␣ subunits of the G i family, including ␣ i , ␣ o , and ␣ z , two distinct acylation events occur. N-Myristoylation takes place at the amino terminus and represents the attachment of a myristate through an amide bond to Gly 2 following cleavage of the initiator methionine (5, 6). Palmitoylation occurs at the adjacent Cys 3 and represents the attachment of a palmitate (primarily) through a thioester bond (7,8). ␣ s , ␣ q , and ␣ 12 family members are palmitoylated but not N-myristoylated (8 -11), whereas ␣ t is N-myristoylated but not palmitoylated (12). The functional consequences of N-myristoylation and palmitoylation have been studied intensively. NMyristoylation plays an important role in the attachment of G i family ␣ subunits to membrane (5, 6), targeting of the subunits to caveolin-enriched membrane domains (13), and interactions with the ␤␥ heterodimer (14) and effectors such as adenylyl cyclase (15). Palmitoylation is relevant to the attachment of G protein ␣ subunits to membrane (10, 16) and interaction of subunits with ␤␥ (17) and RGS proteins (18).The palmitoylation of G protein ␣ subunits has drawn much attention recently, as this modification, unlike N-myristoylation, is reversible and subject to regulation. Several groups have demonstrated that the activation of G s is accompanied by an increased rate of palmitate exchange on ␣ s , probably reflecting depalmitoylation and repalmitoylation of the subunit released from ␤␥ (19 -21)....

Nearly all ␣ subunits of heterotrimeric GTP-binding regulatory proteins (G proteins) are palmitoylated at cysteine residues near the N terminus. A regulated cycle of palmitoylation could provide a mechanism for modulating G protein signaling by affecting protein interactions and localization of the subunit. In the present studies we utilized both Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) 1 transduce signals from cell-surface receptors to intracellular effectors via multiple pathways. The phenomenon of signal transduction is relevant to both normal and abnormal physiological processes, prompting significant advances toward understanding how signal amplitude and duration are regulated at the level of receptor, G protein, and effector. Modulation of signaling has been suggested to occur through direct protein interactions (1) and by dynamic modifications that include phosphorylation (2, 3) and palmitoylation (4 -9).Palmitoylation is a reversible post-translational modification that links the fatty acid palmitate to cysteine residues by a thioester bond. Nearly all ␣ subunits of the four G protein families, G s , G i , G q , and G 12 , are palmitoylated at cysteine residues near the N terminus (10 -14). G i family ␣ subunits, which are palmitoylated at Cys 3 , are also co-translationally N-myristoylated at Gly 2 via an irreversible amide bond (9). Protein-bound palmitate has widespread functional significance, influencing protein interactions and membrane localization for G protein ␣ subunits and other proteins including G protein-coupled receptors and kinases (15-17), Ras proteins (18 -20), nonreceptor tyrosine kinases (21-23), and endothelial nitric-oxide synthase (24, 25).For the G i family ␣ subunits, palmitoylation decreases the affinity of ␣ i and ␣ z for the regulators of G protein signaling (RGS) proteins RGS4, GAIP, and G z GAP, and decreases the maximal rate of GTP hydrolysis of the ␣ subunit-RGS protein complex (26). Cys 3 mutations, which abrogate palmitoylation, have been reported variously to enhance D 2 receptor-mediated inhibition of adenylyl cyclase by ␣ z (27), to have no effect on D 2 receptor-mediated activation of mitogen-activated protein kinase by ␣ z (28), and to inhibit coupling of the ␣ 2A -adrenoreceptor to ␣ i (29). Cys 3 mutations also decrease the extent of subunit association with membrane (12, 27), which for ␣ i (30) and ␣ z (28) is rescued by overexpression of ␤␥. It has been proposed that palmitoylation of ␣ subunits maintains membrane association as part of a dual signal (28) bilayer-trapping mechanism (31) similar to that proposed for acylated nonreceptor tyrosine kinases (32) and Ras proteins (20). Furthermore, palmitoylation may maintain ␣ subunits specifically at the plasma membrane and quite possibly in specific subdomains (33, 34) because palmitoylation-deficient ␣ z mutants mistarget to internal membranes (28), as do nonpalmitoylated Gpa1 (yeast ␣ subunit) (35), fyn (22),.Regulated palmitoylation could provide a mechanism for modulating G p...

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