The helical domain of a G protein α subunit is a regulator of its effector

The α subunit (G��) of heterotrimeric G proteins is a major determinant of signaling selectivity. The G�� structure essentially comprises a GTPase “Ras-like” domain (RasD) and a unique α-helical domain (HD). We used the vertebrate phototransduction model to test for potential functions of HD and found that the HD of the retinal transducin G�� (G��t) and the closely related gustducin (G��g), but not G��i1, G��s, or G��q synergistically enhance guanosine 5′-γ[-thio]triphosphate bound G��t (G��tGTPγS) activation of bovine rod cGMP phosphodiesterase (PDE). In addition, both HDt and HDg, but not HDi1, HDs, or HDq attenuate the trypsin-activated PDE. G��tGDP and HDt attenuation of trypsin-activated PDE saturate with similar affinities and to an identical 38% of initial activity. These data suggest that interaction of intact G��t with the PDE catalytic core may be caused by the HD moiety, and they indicate an independent site(s) for the HD moiety of G��t within the PDE catalytic core in addition to the sites for the inhibitory Pγ subunits. The HD moiety of G��tGDP is an attenuator of the activated catalytic core, whereas in the presence of activated G��tGTPγS the independently expressed HDt is a potent synergist. Rhodopsin catalysis of G��t activation enhances the PDE activation produced by subsaturating levels of G��t, suggesting a HD-moiety synergism from a transient conformation of G��t. These results establish HD-selective regulations of vertebrate retinal PDE, and they provide evidence demonstrating that the HD is a modulatory domain. We suggest that the HD works in concert with the RasD, enhancing the efficiency of G protein signaling.

Inhibition of regulator of G protein signaling function by two mutant RGS4 proteins

Regulators of G protein signaling (RGS) proteins limit the lifetime of activated (GTP-bound) heterotrimeric G protein α subunits by acting as GTPase-activating proteins (GAPs). Mutation of two residues in RGS4, which, based on the crystal structure of RGS4 complexed with Giα1-GDP-AlF4−, directly contact Giα1 (N88 and L159), essentially abolished RGS4 binding and GAP activity. Mutation of another contact residue (S164) partially inhibited both binding and GAP activity. Two other mutations, one of a contact residue (R167M/A) and the other an adjacent residue (F168A), also significantly reduced RGS4 binding to Giα1-GDP-AlF4−, but in addition redirected RGS4 binding toward the GTPγS-bound form. These two mutant proteins had severely impaired GAP activity, but in contrast to the others behaved as RGS antagonists in GAP and in vivo signaling assays. Overall, these results are consistent with the hypothesis that the predominant role of RGS proteins is to stabilize the transition state for GTP hydrolysis. In addition, mutant RGS proteins can be created with an altered binding preference for the Giα-GTP conformation, suggesting that efficient RGS antagonists can be developed.

Organization of G Proteins and Adenylyl Cyclase at the Plasma Membrane

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein α and β subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein α subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.

Low- and high-level transgenic expression of β2-adrenergic receptors differentially affect cardiac hypertrophy and function in G��q-overexpressing mice

Transgenic overexpression of G��q in the heart triggers events leading to a phenotype of eccentric hypertrophy, depressed ventricular function, marked expression of hypertrophy-associated genes, and depressed β-adrenergic receptor (βAR) function. The role of βAR dysfunction in the development of this failure phenotype was delineated by transgenic coexpression of the carboxyl terminus of the βAR kinase (βARK), which acts to inhibit the kinase, or concomitant overexpression of the β2AR at low (≈30-fold, G��q/β2ARL), moderate (≈140-fold, G��q/β2ARM), and high (≈1,000-fold, G��q/β2ARH) levels above background βAR density. Expression of the βARK inhibitor had no effect on the phenotype, consistent with the lack of increased βARK levels in G��q mice. In marked contrast, G��q/β2ARL mice displayed rescue of hypertrophy and resting ventricular function and decreased cardiac expression of atrial natriuretic factor and α-skeletal actin mRNA. These effects occurred in the absence of any improvement in basal or agonist-stimulated adenylyl cyclase (AC) activities in crude cardiac membranes, although restoration of a compartmentalized β2AR/AC signal cannot be excluded. Higher expression of receptors in G��q/β2ARM mice resulted in salvage of AC activity, but hypertrophy, ventricular function, and expression of fetal genes were unaffected or worsened. With ≈1,000-fold overexpression, the majority of G��q/β2ARH mice died with cardiomegaly at 5 weeks. Thus, although it appears that excessive, uncontrolled, or generalized augmentation of βAR signaling is deleterious in heart failure, selective enhancement by overexpressing the β2AR subtype to limited levels restores not only ventricular function but also reverses cardiac hypertrophy.

Fidelity of G protein β-subunit association by the G protein γ-subunit-like domains of RGS6, RGS7, and RGS11

Several regulators of G protein signaling (RGS) proteins contain a G protein γ-subunit-like (GGL) domain, which, as we have shown, binds to G��5 subunits. Here, we extend our original findings by describing another GGL-domain-containing RGS, human RGS6. When RGS6 is coexpressed with different G�� subunits, only RGS6 and G��5 interact. The expression of mRNA for RGS6 and G��5 in human tissues overlaps. Predictions of α-helical and coiled-coil character within GGL domains, coupled with measurements of G�� binding by GGL domain mutants, support the contention that G��-like regions within RGS proteins interact with G��5 subunits in a fashion comparable to conventional G��/G�� pairings. Mutation of the highly conserved Phe-61 residue of G��2 to tryptophan, the residue present in all GGL domains, increases the stability of the G��5/G��2 heterodimer, highlighting the importance of this residue to GGL/G��5 association.

Structure-based design of selective and potent G quadruplex-mediated telomerase inhibitors

The telomerase enzyme is a potential therapeutic target in many human cancers. A series of potent inhibitors has been designed by computer modeling, which exploit the unique structural features of quadruplex DNA. These 3,6,9-trisubstituted acridine inhibitors are predicted to interact selectively with the human DNA quadruplex structure, as a means of specifically inhibiting the action of human telomerase in extending the length of single-stranded telomeric DNA. The anilino substituent at the 9-position of the acridine chromophore is predicted to lie in a third groove of the quadruplex. Calculated relative binding energies predict enhanced selectivity compared with earlier 3,6-disubstituted compounds, as a result of this substituent. The ranking order of energies is in accord with equilibrium binding constants for quadruplex measured by surface plasmon resonance techniques, which also show reduced duplex binding compared with the disubstituted compounds. The 3,6,9-trisubstututed acridines have potent in vitro inhibitory activity against human telomerase, with EC50 values of up to 60 nM.