The Nucleotide Exchange Factor Ric-8A is a Chaperone for the Conformationally Dynamic Nucleotide-Free State of G Alpha I1

Heterotrimeric G protein alpha subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of G alpha, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class G alpha subunits. Ric-8A binds to G alpha.GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free G alpha i1 is stable, but dissociates upon binding of GTP to G alpha i1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from G alpha i1, experiments were conducted to characterize the physical state of nucleotide-free G alpha i1 (hereafter referred to as G alpha i1[]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free G alpha i1 is more accessible to trypsinolysis than G alpha i1.GDP, but less so than G alpha i1[] alone. The TROSY-HSQC spectrum of [N-15]G alpha i1[] bound to Ric-8A shows considerable loss of peak intensity relative to that of [N-15]G alpha i1.GDP. Hydrogen-deuterium exchange in G alpha i1[] bound to Ric-8A is 1.5-fold more extensive than in G alpha i1.GDP. Differential scanning calorimetry shows that both Ric-8A and G alpha i1.GDP undergo cooperative, irreversible unfolding transitions at 47 degrees and 52 degrees, respectively, while nucleotide-free G alpha i1 shows a broad, weak transition near 35 degrees. The unfolding transition for Ric-8A: G alpha i1[] is complex, with a broad transition that peaks at 50 degrees, suggesting that both Ric-8A and G alpha i1[] are stabilized within the complex, relative to their respective free states. The C-terminus of G alpha i1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of G alpha

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

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 proteincoupled receptor kinase 2, a known Gαq modulator, led to a complete abrogation of ERK5 stimulation. Finally, we reveal thatGαq/ 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.Ministerio de Educación y Ciencia (SAF2011-23800, SAF2014-55511-R), Fundación Ramón Areces, The Cardiovascular Diseases Network of Ministerio Sanidad y Consumo-Instituto Carlos III (RD12/0042/0012), Comunidad de Madrid (S-2011/BMD-2332), and Instituto de Salud Carlos III (PI11/00126, PI14/00201) (to F. M. and C. R.). This work was also supported in part by the NIGMS, National Institutes of Health Grant R01-GM088242 (to G. T.), the Canadian Institutes of Health Research (CIHR) (MOP-GMX-231013) (to S. M.), an EMBO Short Fellowship (to G. S. F.), and Fondo Europeo de Desarrollo Regional (FEDER, European Union)Peer Reviewe

Interdicting G q Activation in Airway Disease by Receptor-Dependent and Receptor-Independent Mechanisms

ABSTRACT Ga q bg heterotrimer (G q ), an important mediator in the pathology of airway disease, plays a central role in bronchoconstriction and airway remodeling, including airway smooth muscle growth and inflammation. Current therapeutic strategies to treat airway disease include the use of muscarinic and leukotriene receptor antagonists; however, these pharmaceuticals demonstrate a limited clinical efficacy as multiple G q -coupled receptor subtypes contribute to these pathologies. Thus, broadly inhibiting the activation of G q may be an advantageous therapeutic approach. Here, we investigated the effects of broadly inhibiting G q activation in vitro and ex vivo using receptor-dependent and receptor-independent strategies. P4pal-10 is a protease activated receptor 4-derived pepducin that exhibits efficacy toward multiple G q -coupled receptors. Mechanistic studies demonstrated that P4pal-10 selectively inhibits all G protein coupling to several G q -coupled receptors, including protease activated receptor 1, muscarinic acetylcholine M3, and histamine H1 receptors, while demonstrating no direct effect on G q . We also evaluated the ability of FR900359, also known as UBO-QIC, to directly inhibit G q activation. FR900359 inhibited spontaneous Ga q nucleotide exchange, while having little effect on Ga s bg, Ga i bg, or Ga 12/13 bg heterotrimer activity. Both P4pal-10 and FR900359 inhibited G qmediated intracellular signaling and primary human airway smooth muscle growth, whereas only FR900359 effectively interdicted agonist-promoted airway contraction in human precision cut lung slices. These studies serve as a proof of concept that the broad-based inhibition of G q activation may be a useful therapeutic approach to treat multiple common pathologies of airway disease

Genetic Determination and Linkage Mapping of Plasmodium falciparum Malaria Related Traits in Senegal

Plasmodium falciparum malaria episodes may vary considerably in their severity and clinical manifestations. There is good evidence that host genetic factors contribute to this variability. To date, most genetic studies aiming at the identification of these genes have used a case/control study design for severe malaria, exploring specific candidate genes. Here, we performed a family-based genetic study of falciparum malaria related phenotypes in two independent longitudinal survey cohorts, as a first step towards the identification of genes and mechanisms involved in the outcome of infection. We studied two Senegalese villages, Dielmo and Ndiop that differ in ethnicity, malaria transmission and endemicity. We performed genome-scan linkage analysis of several malaria-related phenotypes both during clinical attacks and asymptomatic infection. We show evidence for a strong genetic contribution to both the number of clinical falciparum malaria attacks and the asymptomatic parasite density. The asymptomatic parasite density showed linkage to chromosome 5q31 (LOD = 2.26, empirical p = 0.0014, Dielmo), confirming previous findings in other studies. Suggestive linkage values were also obtained at three additional chromosome regions: the number of clinical malaria attacks on chromosome 5p15 (LOD = 2.57, empirical p = 0.001, Dielmo) and 13q13 (LOD = 2.37, empirical p = 0.0014 Dielmo), and the maximum parasite density during asymptomatic infection on chromosome 12q21 (LOD = 3.1, empirical p<10−4, Ndiop). While regions of linkage show little overlap with genes known to be involved in severe malaria, the four regions appear to overlap with regions linked to asthma or atopy related traits, suggesting that common immune related pathways may be involved

Ric-8 Proteins Are Molecular Chaperones Required For The Biogenesis G Protein Alpha Subunits

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2012.Ric-8A (resistance to inhibitors of cholinesterase 8A) and Ric-8B are guanine nucleotide exchange factors that enhance different heterotrimeric G protein signaling pathways by unknown mechanisms. Because transgenic disruption of Ric-8A or Ric-8B in mice caused early embryonic lethality, we derived viable Ric-8A– or Ric-8B–deleted embryonic stem (ES) cell lines from blastocysts of these mice. We observed pleiotropic G protein signaling defects in Ric-8A-/- ES cells, which resulted from reduced steady-state amounts of Gαi, Gαq, and Gα13 proteins to <5% of those of wild-type cells. Only the amount of Gαs was reduced substantially in Ric-8B-/- cells. The plasma membrane residence of G proteins persisted in the absence of Ric-8 but was markedly reduced compared to that in wild-type cells. Endogenous Gαi and Gαq were efficiently translated in Ric-8A-/- cells but integrated into endomembranes poorly; however, the reduced amounts of Gα subunits that reached the membrane still bound to nascent Gβγ. Interestingly, the Gαi and Gαq proteins that remained soluble in the Ric-8A- /- cells, had a higher apparent molecular weight, suggestive towards the presence of a putative post translational modification (PTM) that is revealed when Ric-8A is absent. In addition, Gαi, Gαq, and Gβ1 proteins exhibited accelerated rates of degradation in Ric- 8A-/- cells compared to those in wild-type cells. Together, these data suggest that Ric-8 proteins are molecular chaperones required for the initial association of nascent Gα subunits with cellular membranes. From further investigation of the slower migrating Gα proteins we concluded that in the absence of Ric-8 proteins, newly synthesized Gα proteins are not properly folded and become resistant to an artifactual proteolytic event that occurs by cellular proteases. In addition, we found that Ric-8A is natively phosphorylation in mammalian cells, however some site directed mutations of predicted phosphorylated sites resulted in full rescue of G protein expression while others generated a Ric-8A protein that was unstable when expressed in ES or insect cells. We believe that phosphorylation or dephosphorylation of Ric-8A has a functional purpose that can reveal further mechanistic information in regards to the folding and initial trafficking of Gα proteins, as well as the discovery of cellular Ric-8 activators

The Molecular Chaperoning and GEF Functions of Ric-8 in Heterotrimeric G protein Signaling

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2013.Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a non-receptor guanine nucleotide exchange factor (GEF) that stimulates the exchange of guanine nucleotide (GDPàGTP) for heterotrimeric G protein alpha subunits (Gα). In cells, active G proteins (Gα-GTP) alter numerous signaling pathways to provoke biological responses. Overexpression of Ric-8B enhances G protein signaling in cell culture systems, and it has long been postulated, but never been shown to be a Gα GEF. We found that Ric-8B proteins were Gαs GEFs, but the catalysis of nucleotide exchange was GTP dependent. This phenomenon is unusual for a GEF. At low GTP levels, Ric-8B potently inhibited Gαs steady state GTP consumption (catalysis), but at high GTP levels, Ric-8B proteins are Gαs activators. We conclude that at cellular GTP levels, Ric-8B catalyzes nucleotide exchange for Gαs in a forward direction to produce Gα-GTP in cells. Although Ric-8 is a Gα activator in vitro, the actual function of Ric-8 GEF activity in cells was not known. We found that co-overexpression of Ric-8 and Gα baculoviruses in insect cells dramatically enhanced our production of functional purified Gα proteins. With this method, unprecedented quantities of representatives of each Gα class were obtained. We showed that Ric-8 proteins were molecular chaperones that direct nascent G protein α subunits membrane association. To determine the function of Ric-8 in G protein biosynthesis, cell free translation systems were used to ascertain the effect of Ric- 8 on G protein folding. In Ric-8A immuno-depleted rabbit reticulocyte lysate (RRL), Gα proteins were not able to adopt the active conformation, indicating an improper Gα fold. Addition of recombinant Ric-8A protein to Ric-8A-depleted RRL or wheat germ extract enhanced the production of functional Gα. This study shows that Ric-8 positive influence of G protein signaling is a consequence of Ric-8 folding newly synthesized Gα subunits to produce functional G proteins. Ric-8 GEF activity might be an in vitro phenomenon related to the ability of Ric-8 to mediate the first event of GTP binding to newly folded Gα. My work has prompted studies to identify additional cellular chaperones that might work with Ric-8A during G protein biosynthesis/folding

Ric-8A Deletion and Phorbol Ester Suppresses Tumorigenesis in a Mouse Model of GNAQQ209L -driven Melanoma

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2016.Activating mutations at amino acid residues Q209 or R183 of heterotrimeric G protein α subunit GNAQ/11 are found in ~90% of human uveal melanomas. Tumordriving capacity of Gαq/11Q209/R183 mutant has been demonstrated in mouse models. However, there are no experimental or clinical inhibitors of mutated G proteins. This prompted us to investigate Ric-8A, a selective molecular chaperone for Gαq/i/13 subunits, as a means to reduce the functional abundance of oncogenic Gαq/11 and attenuate downstream oncogenic signaling. To address this, we created a conditional- Ric-8A knockout mouse and subsequently derived a mouse melanocyte cell lines with tamoxifen-inducible Ric-8A knockout potential. The derived cell line is non-tumorigenic in immune-deficient mice and is dependent on 12-O-tetradecanoylphorbol-13-acetate (TPA, phorbol ester) for proliferation regardless of Ric-8A expression. To determine the effect of Ric-8A deletion on the abundance and melanoma-driving potential of Gαq-Q209L, we stably express GNAQQ209L oncogene in inducible Ric-8A-knockout melanocyte cell lines. Expression of GNAQQ209L in the melanocyte cell line drove TPA-independent cell growth in vitro and melanoma tumor growth from subcutaneous cell graft in immune-deficient mice. Genetic ablation of Ric-8A in GNAQQ209L melanocyte cell line attenuated TPAindependent growth by reducing the levels of Gαq-Q209L oncoprotein. Melanoma tumorigenesis driven by primary and secondary GNAQQ209L cell grafts was substantially attenuated by Ric-8A deletion prior to cell grafting. Deletion of endogenous Ric-8A Floxed allele in GNAQQ209L cell graft in vivo attenuated tumor progression. However, expression of RIC-8A transgene rescued the tumor growth-inhibitory effect of endogenous Ric-8A deletion. Interestingly, TPA-treated GNAQQ209L cells also had dramatic decrease in Gαq-Q209L abundance without any change in GNAQQ209L transcript levels. The TPA-treatment of cultured GNAQQ209L melanocytes or host mice grafted with GNAQQ209L melanocytes blocked tumorigenic capacity. Our work identified Ric-8A inhibition and phorbol ester treatment as two novel approaches to attenuate uveal melanoma by reduction of Gαq/11-Q209L oncoprotein abundance

Activation of Adhesion G Protein-coupled Receptors by a Tethered Agonist: Mechanism of Action and Pharmacological Modulation

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2016.The recently defined adhesion G protein-coupled receptors (adhesion GPCRs) are a poorly characterized GPCR family with seven transmembrane topology and very large extracellular domains harboring numerous adhesion modules. These orphan receptors have profound therapeutic potential, with established roles in oncogenesis and metastasis. However, mechanistic understanding of adhesion GPCR activation and intracellular signaling is limited. Hence, efforts to discover pharmacological modulators for these proteins have been stunted. We have uncovered a common mechanism of activation that may apply to all 33 receptors. aGPCRs have an evolutionarily conserved GPCR Autoproteolysis Inducing (GAIN) domain that divides the receptor into two discrete portions: the extracellular domain (ECD) and the 7 transmembrane domain (7TM). The ECD and 7TM are noncovalently bound at the plasma membrane, positioned for activation. It is believed that extracellular matrix proteins bind the ECDs of these proteins, eliciting a removal/rearrangement. We discovered that for G protein receptors 56 and 110, the ECD sequesters an ~20 amino acid tethered agonist covalently bound to the 7TM and that upon ECD removal, the 7TM directly activates heterotrimeric G proteins. Using our established [35S]-GTPγS activation assay, we directly measured receptormediated G protein activation, proving that these receptors are bona fide GPCRs and defining the subtypes of G proteins to which GPRs 56 and 110 will couple. Having established the adhesion GPCR mechanism of activation, we performed luciferase gene reporter assays adapted for high throughput screening to identify inhibitors of the active (tethered agonist bound) GPR56 7TM receptor and activators of a low-activity (truncated tethered agonist) GPR56 7TM receptor. The isoflavonoid, dihydromunduletone, was the most efficacious GPR56 inhibitor while the natural product isolated from the Indian Neem tree, 3-alphaacetoxydihydrodeoxygedunin, was an efficacious GPR56 activator. Both compounds were able to respectively inhibit and activate other adhesion GPCRs including the related GPR114 but could not inhibit or activate other non-adhesion GPCRs, including the β2 adrenergic receptor and the M3 muscarinic receptor. Collectively, we have uncovered the adhesion GPCR activation mechanism and used it to identify an aGPCR inhibitor and an aGPCR activator that may be used for lead optimization and eventual therapeutic use