Catalina Ribas

Receptor-independent Activators of Heterotrimeric G-protein Signaling Pathways

Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane span motif. Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins. As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae. We identified two mammalian proteins, AGS2 and AGS3 (activators of G-proteinsignaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. β-galactosidase reporter assays in yeast strains expressing different Gα subunits (Gpa1, Gsα, Giα2 (Gpa1(1–41)), Giα3(Gpa1(1–41)), Gα16(Gpa1(1–41))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the Giα2 and Giα3genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gβγ, whereas AGS3 bound Gα and exhibited a preference for GαGDP versus GαGTPγS. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled receptor. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways. AGS2 and AGS3 may also serve as novel binding partners for Gα and Gβγ that allow the subunits to subserve functions that do not require initial heterotrimer formation.

Activation of heterotrimeric G-protein signaling by a ras-related protein. Implications for signal integration.

Utilizing a functional screen in the yeastSaccharomyces cerevisiae we identified mammalian proteins that activate heterotrimeric G-protein signaling pathways in a receptor-independent fashion. One of the identified activators, termed AGS1 (for activator of G-proteinsignaling), is a human Ras-related G-protein that defines a distinct subgroup of the Ras superfamily. Expression of AGS1 in yeast and in mammalian cells results in specific activation of Gαi/Gαo heterotrimeric signaling pathways. In addition, the in vivo and in vitroproperties of AGS1 are consistent with it functioning as a direct guanine nucleotide exchange factor for Gαi/Gαo. AGS1 thus presents a unique mechanism for signal integration via heterotrimeric G-protein signaling pathways.

Characterization of a G-protein Activator in the Neuroblastoma-Glioma Cell Hybrid NG108-15

Purified bovine brain G-protein was used in a solution phase assay to identify membrane-associated proteins that influenced the activation of heterotrimeric G-proteins. Detergent-solubilized membrane extracts from the neuroblastoma-glioma cell hybrid NG108-15, but not the parent C6B4 glioma cell line, increased [35S]GTPγS binding to purified G-protein by ∼460%. The G-protein activator was heat-sensitive, and the magnitude of its action was related to the amount of extract protein. The biophysical and biochemical properties of the G-protein activator were determined using DEAE ion exchange chromatography, gel filtration, and a lectin affinity matrix. In the presence of added GDP (1 μM), the enriched G-protein activator increased the initial rate of [35S]GTPγS binding to brain G-protein by up to 4-fold. In the absence of added GDP, the G-protein activator elicited an initial burst in [35S]GTPγS binding to brain G-protein within the first 30 s, after which the rate of nucleotide binding to G-protein was similar in the absence or presence of the G-protein activator. The stimulation of nucleotide binding to brain G-protein by the activator was also observed after resolution of Gα from Gβγ. The G-protein activator was distinct from other proteins (neuromodulin, tubulin, and β-amyloid precursor protein) that influence nucleotide binding to G-protein, indicating the existence of a novel signal accelerator.

Gαq Acts as an Adaptor Protein in Protein Kinase Cζ (PKCζ)-mediated ERK5 Activation by G Protein-coupled Receptors (GPCR)

Gq-coupled G protein-coupled receptors (GPCR) mediate the actions of a variety of messengers that are key regulators of different cellular functions. These receptors can regulate a highly interconnected network of biochemical routes that control the activity of several members of the mitogen-activated protein kinase (MAPK) family. The ERK5 MAPK has been shown to be activated by Gq-coupled GPCR via unknown mechanisms. We find that the atypical protein kinase C (PKCζ), previously reported to interact with the ERK5 activator MEK5 and to be involved in epidermal growth factor-mediated ERK5 stimulation, plays a crucial role in the activation of the ERK5 pathway by Gq-coupled GPCR. Stimulation of ERK5 by Gq-coupled GPCR is abolished upon pharmacological inhibition of PKCζ as well as in embryonic fibroblasts obtained from PKCζ-deficient mice. Both PKCζ and MEK5 associate to Gαq upon activation of GPCR, thus forming a ternary complex that seems essential for the activation of ERK5. These data put forward a novel function of Gαq as a scaffold protein involved in the modulation of the ERK5 cascade by GPCR that could be relevant in Gq-mediated physiological functions.

Gαq acts as an adaptor protein in PKCζ-mediated ERK5 activation by GPCR

Gq-coupled G protein-coupled receptors (GPCR) mediate the actions of a variety of messengers that are key regulators of different cellular functions. These receptors can regulate a highly interconnected network of biochemical routes that control the activity of several members of the mitogen-activated protein kinase (MAPK) family. The ERK5 MAPK has been shown to be activated by Gq-coupled GPCR via unknown mechanisms. We find that the atypical protein kinase C (PKCζ), previously reported to interact with the ERK5 activator MEK5 and to be involved in epidermal growth factor-mediated ERK5 stimulation, plays a crucial role in the activation of the ERK5 pathway by Gq-coupled GPCR. Stimulation of ERK5 by Gq-coupled GPCR is abolished upon pharmacological inhibition of PKCζ as well as in embryonic fibroblasts obtained from PKCζ-deficient mice. Both PKCζ and MEK5 associate to Gαq upon activation of GPCR, thus forming a ternary complex that seems essential for the activation of ERK5. These data put forward a novel function of Gαq as a scaffold protein involved in the modulation of the ERK5 cascade by GPCR that could be relevant in Gq-mediated physiological functions.

Protein Kinase C (PKC)ζ-mediated Gαq Stimulation of ERK5 Protein Pathway in Cardiomyocytes and Cardiac Fibroblasts

Background: We have recently described that Gαq acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5. Results: Our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts. Conclusion: This novel signaling axis plays a key role in cardiac hypertrophy programs. Significance: The Gαq/PKCζ/ERK5 pathway would be active in such pathological settings, providing new therapeutic targets. Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gαq-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gαq acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKCζ-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the Gαq/PKCζ/ERK5 signaling axis.

Protein kinase C ζ interacts with a novel binding region of Gαq to act as functional effector protein

Heterotrimeric G proteins play an essential role in the initiation of G protein-coupled receptor (GPCR) signaling through specific interactions with a variety of cellular effectors. We have recently reported that GPCR activation promotes a direct interaction between Gαq and protein kinase C ζ (PKCζ), leading to the stimulation of the ERK5 pathway independent of the canonical effector PLCβ. We report herein that the activation-dependent Gαq/PKCζ complex involves the basic PB1-type II domain of PKCζ and a novel interaction module in Gαq different from the classical effector-binding site. Point mutations in this Gαq region completely abrogate ERK5 phosphorylation, indicating that Gαq/PKCζ association is required for the activation of the pathway. Indeed, PKCζ was demonstrated to directly bind ERK5 thus acting as a scaffold between Gαq and ERK5 upon GPCR activation. The inhibition of these protein complexes by G protein-coupled receptor kinase 2, a known Gαq modulator, led to a complete abrogation of ERK5 stimulation. Finally, we reveal that Gαq/PKCζ complexes link Gαq to apoptotic cell death pathways. Our data suggest that the interaction between this novel region in Gαq and the effector PKCζ is a key event in Gαq signaling.

Chapter 16:The Complex Role of G Protein-coupled Receptor Kinase 2 (GRK2) in Cell Signalling: Beyond GPCR Desensitization

G protein-coupled receptor kinases (GRKs) were initially identified as key players of G protein-coupled receptors (GPCR) desensitization by promoting agonist-induced receptor phosphorylation and subsequent arrestin binding. However, cumulative evidence is putting forward novel roles for GRKs, particularly for the ubiquitous GRK2 isoform. GRK2 can phosphorylate a growing number of non-GPCR substrates and associate with a variety of proteins related to signal transduction, thus suggesting that this kinase could also have diverse, unforeseen ‘effector’ functions. In fact, the increasing complexity of the GRK2 ‘interactome’ suggest that this kinase plays a central, integrative role in signal transduction cascades. Ongoing research is identifying how specific GRK2 interactomes are orchestrated in a stimulus, cell type or context-specific way. This will help to understand how alterations in GRK2 levels or functionality participate in the onset and development of several cardiovascular and inflammatory diseases, and of some types of tumors.