Heterotrimeric G proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP bound state and an active, GTP bound state. Under basal conditions, G proteins exist in the inactive, GDP bound state; thus, nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide state-dependent Galpha binding peptides. Herein, we report a GDP-selective Galpha binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Galpha(i) subunits. Structural determination of the Galpha(i1)/peptide complex reveals unique changes in the Galpha switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Galpha subunits adopt a conformation suitable for nucleotide exchange.

G protein-coupled inwardly rectifying potassium (GIRK) channels can be activated or inhibited by different classes of receptors, suggesting a role for G proteins in determining signaling specificity. Because G protein ␤␥ subunits containing either ␤1 or ␤2 with multiple G␥ subunits activate GIRK channels, we hypothesized that specificity might be imparted by ␤3, ␤4, or ␤5 subunits. We used a transfection assay in cell lines expressing GIRK channels to examine effects of dimers containing these G␤ subunits. Inwardly rectifying K ؉ currents were increased in cells expressing ␤3 or ␤4, with either ␥2 or ␥11. Purified, recombinant ␤3␥2 and ␤4␥2 bound directly to glutathione-S-transferase fusion proteins containing N-or C-terminal cytoplasmic domains of GIRK1 and GIRK4, indicating that ␤3 and ␤4, like ␤1, form dimers that bind to and activate GIRK channels. By contrast, ␤5-containing dimers inhibited GIRK channel currents. This inhibitory effect was obtained with either ␤5␥2 or ␤5␥11, was observed with either GIRK1,4 or GIRK1,2 channels, and was evident in the context of either basal or agonist-induced currents, both of which were mediated by endogenous G␤␥ subunits. In cotransfection assays, ␤5␥2 suppressed ␤1␥2-activated GIRK currents in a dose-dependent manner consistent with competitive inhibition. Moreover, we found that ␤5␥2 could bind to the same GIRK channel cytoplasmic domains as other, activating G␤␥ subunits. Thus, ␤5-containing dimers inhibit G␤␥-stimulated GIRK channels, perhaps by directly binding to the channels. This suggests that ␤5-containing dimers could act as competitive antagonists of other G␤␥ dimers on GIRK channels. P otassium channels that are active near resting membrane potentials are key determinants of cellular excitability. The G protein-coupled inwardly rectifying K ϩ (GIRK; Kir3.x) channels are particularly interesting in that they are differentially regulated by receptors that couple to different classes of heterotrimeric G proteins: GIRK channels are activated by receptors that couple to G␣i͞o and inhibited by receptors that couple to G␣q (1, 2). This dual up-and down-regulation of GIRK channels by different receptor classes has been described in atrial cells (3), aminergic brainstem neurons (4, 5), and enteric neurons of the peripheral nervous system (6).Mechanisms underlying inhibition of GIRK channels are not well understood. By contrast, the characteristics of receptormediated activation of GIRK channels have been worked out in detail. It is now clear that G␤␥ subunits liberated from G protein heterotrimers bind directly to GIRK channels to enhance channel activity (reviewed in refs. 1 and 2). This mechanism raises an interesting conundrum: If all G protein-coupled receptors release G␤␥ subunits when activated and all G␤␥ subunits tested to date activate GIRK channels (7), how is signaling specificity obtained such that different classes of receptor can activate or inhibit GIRK channels?One possibility is that specificity derives from associations of different receptors with par...

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Heterotrimeric G proteins relay information between cell surface receptors and effector molecules in diverse signaling pathways to mediate critical cellular processes in both physiologic and pathologic conditions. Multiple isoforms of each of the three G protein subunits yield enormous structural and functional diversity. G proteins are thus obvious molecular targets for the therapeutic manipulation of signaling pathways. Their ubiquity among a vast array of G protein-coupled receptor pathways, however, may at first seem to threaten the attractiveness of G proteins as drug targets for specific signaling processes; in order for G proteins to be effective targets, some degree of selectivity must be defined and exploited. Although a great deal has been determined about the functional selectivity of G alpha subunits, relatively little is known regarding G betagamma selectivity. In this review, we discuss functional diversity among G betagamma subunits in both receptor coupling and effector activation. The novel functions of G beta(5), in complex with proteins of the GGL domain-containing R7 subfamily of regulators of G protein signaling, are discussed in detail, with specific focus on the potential of the G beta(5)-RGS9-2 pair as a therapeutic target in Parkinson's disease.

GoLoco ('Gα i/o -Loco' interaction) motif proteins have recently been identified as novel GDIs (guanine nucleotide dissociation inhibitors) for heterotrimeric G-protein α subunits. G18 is a member of the mammalian GoLoco-motif gene family and was uncovered by analyses of human and mouse genomes for anonymous open-reading frames. The encoded G18 polypeptide is predicted to contain three 19-amino-acid GoLoco motifs, which have been shown in other proteins to bind Gα subunits and inhibit spontaneous nucleotide release. However, the G18 protein has thus far not been characterized biochemically. Here, we have cloned and expressed the G18 protein and assessed its ability to act as a GDI. G18 is capable of simultaneously binding more than one Gα i1 subunit. In binding assays with the non-hydrolysable GTP analogue guanosine 5 -[γ -thio]triphosphate, G18 exhibits GDI activity, slowing the exchange of GDP for GTP by Gα i1 . Only the first and third GoLoco motifs within G18 are capable of interacting with Gα subunits, and these bind with low micromolar affinity only to Gα i1 in the GDP-bound form, and not to Gα o , Gα q , Gα s or Gα 12 . Mutation of Ala-121 to aspartate in the inactive second GoLoco motif of G18, to restore the signature acidicglutamine-arginine tripeptide that forms critical contacts with Gα and its bound nucleotide [Kimple, Kimple, Betts, Sondek and Siderovski (2002) Nature (London) 416, 878-881], results in gain-of-function with respect to Gα binding and GDI activity.