G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.

Phospholipase C-β (PLCβ) is directly activated by Gαq, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM 3D reconstructions of human full-length PLCβ3 in complex with murine Gαq. The distal CTD forms an extended, monomeric helical bundle consisting of three anti-parallel segments with structural similarity to membrane-binding bin–amphiphysin–Rvs (BAR) domains. Sequence conservation of the distal CTD identifies putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gαq in both the crystal structure and cryo-EM reconstructions. Functional analysis suggests the distal CTD plays roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis.

Activation of certain classes of G protein-coupled receptors (GPCRs) can lead to alterations in the actin cytoskeleton, gene transcription, cell transformation, and other processes that are known to be regulated by Rho family small-molecular-weight GTPases. Although these responses can occur indirectly via cross-talk from canonical heterotrimeric G protein cascades, it has recently been demonstrated that Dbl family Rho guanine nucleotide exchange factors (RhoGEFs) can serve as the direct downstream effectors of heterotrimeric G proteins. Heterotrimeric G␣ 12/13 , G␣ q , and G␤␥ subunits are each now known to directly bind and regulate RhoGEFs. Atomic structures have recently been determined for several of these RhoGEFs and their G protein complexes, providing fresh insight into the molecular mechanisms of signal transduction between GPCRs and small molecular weight G proteins. This review covers what is currently known about the structure, function, and regulation of these recently recognized effectors of heterotrimeric G proteins.Heterotrimeric G proteins are master regulators of cell homeostasis. By coordinating signaling between the ϳ800 G protein-coupled receptors (GPCRs) in the human genome and a relatively small handful of effector enzymes and channels in the cell, they control processes such as muscle contractility, glycogen metabolism, neurotransmission, and the concentration of intracellular ions. Their profound impact on nearly all cellular processes and their therapeutic potential have rendered them one of the most intensely studied signal transduction paradigms at the biochemical and molecular level (Sprang et al., 2007).When heterotrimeric G proteins are in their inactive, GDPbound state, they exist as an inert complex composed of ␣, ␤, and ␥ subunits (G␣␤␥). In this state, they are substrates for activated GPCRs, which catalyze nucleotide exchange on the ␣ subunit (G␣). When bound to GTP, G␣ releases the effector binding surface of the ␤ and ␥ heterodimer (G␤␥) so that both G␣ and G␤␥ can interact with and modulate the activity of specific downstream enzymes and channels. The G␣ subunit has weak guanine nucleotide triphosphatase (GTPase) activity that slowly returns the G protein to its GDP-bound state. G␣⅐GDP then becomes resequestered by G␤␥. Beyond serving as conduits for extracellular signals, heterotrimeric G proteins contribute to the fidelity, duration, and amplitude of GPCR signaling. A given class of heterotrimeric G protein can typically recognize only a subset of GPCRs, and can only interact with one or a few downstream effector targets, ensuring the specificity of signaling from receptor to effector. The rate of GTP hydrolysis on G␣ dictates the length of time that its signal is in play, and this rate -bound conformation. The Ras-like domain is colored cyan, and the ␣-helical domain is gray. The three nucleotide-dependent switch regions (switch I-III) are red. The canonical effector docking site, a shallow canyon formed between switch II and the ␣3 helix, is indicated by the tr...

Gα q directly activates p63RhoGEF and closely related catalytic domains found in Trio and Kalirin, thereby linking G q -coupled receptors to the activation of RhoA. Although the crystal structure of Gα q in complex with the catalytic domains of p63RhoGEF is available, the molecular mechanism of activation has not yet been defined. In this study, we show that membrane translocation does not appear to play a role in Gα q -mediated activation of p63RhoGEF, as it does in some other RhoGEFs. Gα q instead must act allosterically. We next identify specific structural elements in the PH domain that inhibit basal nucleotide exchange activity, and provide evidence that Gα q overcomes this inhibition by altering the conformation of the α6-αN linker that joins the DH and PH domains, a region that forms direct contacts with RhoA. We also identify residues in Gα q that are important for the activation of p63RhoGEF and that contribute to Gα subfamily selectivity, including a critical residue in the Gα q C-terminal helix, and demonstrate the importance of these residues for RhoA activation in living cells.

Phospholipase Cβ (PLCβ) is activated in response to specific extracellular stimuli by direct interactions with the heterotrimeric G protein Gαq. However, the molecular mechanisms underlying this regulation are poorly understood. In this work we present four new structures of PLCβ that capture the enzyme in its basal state and in complex with Gαq. These structures not only provide the first high‐resolution models of the C‐terminal regulatory domain in the context of full‐length PLCβ, but also identify regions important for PLCβ regulation and Gαq binding and activation. Deletion or mutation of these regions has profound consequences in the basal activity of PLCβ, the magnitude of Gαq‐stimulated activation, and the affinity for activated Gαq. Taken together, these structures reveal an allosteric mechanism of PLCβ activation by Gαq and define the role of the C‐terminal regulatory domain in this process. Support for this work was provided by NIH grants HL086865 and HL071818 to J.J.G.T. and an American Heart Association postdoctoral fellowship to A.M.L.