The Epstein-Barr Virus-encoded G Protein-coupled Receptor BILF1 Hetero-oligomerizes with Human CXCR4, Scavenges Gαi Proteins,and Constitutively Impairs CXCR4 Functioning

Cells express distinct G protein-coupled receptor (GPCR) subtypes on their surface, allowing them to react to a corresponding variety of extracellular stimuli. Cross-regulation between different ligand-GPCR pairs is essential to generate appropriate physiological responses. GPCRs can physically affect each other''s functioning by forming heteromeric complexes, whereas cross-regulation between activated GPCRs also occurs through integration of shared intracellular signaling networks. Human herpesviruses utilize virally encoded GPCRs to hijack cellular signaling networks for their own benefit. Previously, we demonstrated that the Epstein-Barr virus-encoded GPCR BILF1 forms heterodimeric complexes with human chemokine receptors. Using a combination of bimolecular complementation and bioluminescence resonance energy transfer approaches, we now show the formation of hetero-oligomeric complexes between this viral GPCR and human CXCR4. BILF1 impaired CXCL12 binding to CXCR4 and, consequently, also CXCL12-induced signaling. In contrast, the G protein uncoupled mutant BILF1-K^3.50A affected CXCL12-induced CXCR4 signaling to a much lesser extent, indicating that BILF1-mediated CXCR4 inhibition is a consequence of its constitutive activity. Co-expression of Gα_i1 with BILF1 and CXCR4 restored CXCL12-induced signaling. Likewise, BILF1 formed heteromers with the human histamine H₄ receptor (H₄R). BILF1 inhibited histamine-induced Gα_i-mediated signaling by H₄R, however, without affecting histamine binding to this receptor. These data indicate that functional cross-regulation of Gα_i-coupled GPCRs by BILF1 is at the level of G proteins, even though these GPCRs are assembled in hetero-oligomeric complexes.     

Assembly and Function of the Regulator of G protein Signaling 14 (RGS14)·H-Ras Signaling Complex in Live Cells Are Regulated by Gαi1 and Gαi-linked G Protein-coupled Receptors

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gα_i/o-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gα_i1/3-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive Gα_i1-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gα_i1-GDP but not active Gα_i1-GTP. Active H-Ras(G/V) interacts with a native RGS14·Gα_i1 complex in brain lysates, and co-expression of RGS14 and Gα_i1 in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gα_i-linked α_2A-adrenergic receptor induces a conformational change in the Gα_i1·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gα_i1-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.     

Intracellular Signaling by the Chemokine Receptor US28 during Human Cytomegalovirus Infection

In patients with impaired cell-mediated immune responses (e.g., lung transplant recipients and AIDS patients), cytomegalovirus (CMV) infection causes severe disease such as pneumonitis. However, although immunocompetency in the host can protect from CMV disease, the virus persists by evading the host immune defenses. A model of CMV infection of the endothelium has been developed in which inflammatory stimuli, such as the CC chemokine RANTES, bind to the endothelial cell surface, stimulating calcium flux during late times of CMV infection. At 96 h postinfection, CMV-infected cells express mRNA of the CMV-encoded CC chemokine receptor US28 but do not express mRNA of other CC chemokine receptors that bind RANTES (CCR1, CCR4, CCR5). Cloning and stable expression of the receptor CMV US28 in human kidney epithelial cells (293 cells) with and without the heterotrimeric G protein α₁₆ indicated that CMV US28 couples to both Gα_i and Gα₁₆ proteins to activate calcium flux in response to the chemokines RANTES and MCP-3. Furthermore, cells that coexpress US28 and Gα₁₆ responded to RANTES stimulation with activation of extracellular signal-regulated kinase, which could be attributed, in part, to specific Gα₁₆ coupling. Thus, through expression of the CC chemokine receptor US28, CMV may utilize resident G proteins of the infected cell to manipulate cellular responses stimulated by chemokines.     

β-Arrestin Recruitment and G Protein Signaling by the Atypical Human Chemokine Decoy Receptor CCX-CKR

Chemokine receptors form a large subfamily of G protein-coupled receptors that predominantly activate heterotrimeric G_i proteins and are involved in immune cell migration. CCX-CKR is an atypical chemokine receptor with high affinity for CCL19, CCL21, and CCL25 chemokines, but is not known to activate intracellular signaling pathways. However, CCX-CKR acts as decoy receptor and efficiently internalizes these chemokines, thereby preventing their interaction with other chemokine receptors, like CCR7 and CCR9. Internalization of fluorescently labeled CCL19 correlated with β-arrestin2-GFP translocation. Moreover, recruitment of β-arrestins to CCX-CKR in response to CCL19, CCL21, and CCL25 was demonstrated using enzyme-fragment complementation and bioluminescence resonance energy transfer methods. To unravel why CCX-CKR is unable to activate G_i signaling, CCX-CKR chimeras were constructed by substituting its intracellular loops with the corresponding CCR7 or CCR9 domains. The signaling properties of chimeric CCX-CKR receptors were characterized using a cAMP-responsive element (CRE)-driven reporter gene assay. Unexpectedly, wild type CCX-CKR and a subset of the chimeras induced an increase in CRE activity in response to CCL19, CCL21, and CCL25 in the presence of the G_i inhibitor pertussis toxin. CCX-CKR signaling to CRE required an intact DRY motif. These data suggest that inactive G_i proteins impair CCX-CKR signaling most likely by hindering the interaction of this receptor with pertussis toxin-insensitive G proteins that transduce signaling to CRE. On the other hand, recruitment of the putative signaling scaffold β-arrestin to CCX-CKR in response to chemokines might allow activation of yet to be identified signal transduction pathways.     

A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells

G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gα_i1, Gα_i2 and Gα_i3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gα_i subunit, and cp173Venus fused to the Gγ₂ subunit as acceptor. The Gα_i FRET biosensors constructs are expressed together with Gβ₁ from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gα_i FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gα_i FRET sensor in single cells upon stimulation of several GPCRs, including the LPA₂, M₃ and BK₂ receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gα_i1, Gα_i2 and Gα_i3 activation will be valuable for live-cell measurements that probe Gα_i activation.     

Gα13 Switch Region 2 Binds to the Talin Head Domain and Activates αIIbβ3 Integrin in Human Platelets

Even though GPCR signaling in human platelets is directly involved in hemostasis and thrombus formation, the sequence of events by which G protein activation leads to αIIbβ3 integrin activation (inside-out signaling) is not clearly defined. We previously demonstrated that a conformationally sensitive domain of one G protein, i.e. Gα₁₃ switch region 1 (Gα₁₃SR1), can directly participate in the platelet inside-out signaling process. Interestingly however, the dependence on Gα₁₃SR1 signaling was limited to PAR1 receptors, and did not involve signaling through other important platelet GPCRs. Based on the limited scope of this involvement, and the known importance of G₁₃ in hemostasis and thrombosis, the present study examined whether signaling through another switch region of G₁₃, i.e. Gα₁₃ switch region 2 (Gα₁₃SR2) may represent a more global mechanism of platelet activation. Using multiple experimental approaches, our results demonstrate that Gα₁₃SR2 forms a bi-molecular complex with the head domain of talin and thereby promotes β3 integrin activation. Moreover, additional studies provided evidence that Gα₁₃SR2 is not constitutively associated with talin in unactivated platelets, but becomes bound to talin in response to elevated intraplatelet calcium levels. Collectively, these findings provide evidence for a novel paradigm of inside-out signaling in platelets, whereby β3 integrin activation involves the direct binding of the talin head domain to the switch region 2 sequence of the Gα₁₃ subunit.     

Differentiation of Human T Cells Alters Their Repertoire of G Protein α-Subunits

Because T cell differentiation leads to an expanded repertoire of chemokine receptors, a subgroup of G protein-coupled receptors, we hypothesized that the repertoire of G proteins might be altered in parallel. We analyzed the abundance of mRNA and/or protein of six G protein α-subunits in human CD4⁺ and CD8⁺ T cell subsets from blood. Although most G protein α-subunits were similarly expressed in all subsets, the abundance of Gα_o, a protein not previously described in hematopoietic cells, was much higher in memory versus naive cells. Consistent with these data, activation of naive CD4⁺ T cells in vitro significantly increased the abundance of Gα_o in cells stimulated under nonpolarizing or T_H17 (but not T_H1 or T_H2)-polarizing conditions. In functional studies, the use of a chimeric G protein α-subunit, Gα_qo5, demonstrated that chemokine receptors could couple to Gα_o-containing G proteins. We also found that Gα_i1, another α-subunit not described previously in leukocytes, was expressed in naive T cells but virtually absent from memory subsets. Corresponding to their patterns of expression, siRNA-mediated knockdown of Gα_o in memory (but not naive) and Gα_i1 in naive (but not memory) CD4⁺ T cells inhibited chemokine-dependent migration. Moreover, although even in Gα_o- and Gα_i1-expressing cells mRNAs of these α-subunits were much less abundant than Gα_i2 or Gα_i3, knockdown of any of these subunits impaired chemokine receptor-mediated migration similarly. Together, our data reveal a change in the repertoire of Gα_i/o subunits during T cell differentiation and suggest functional equivalence among Gα_i/o subunits irrespective of their relative abundance.     

Signaling through G protein coupled receptors

Heterotrimeric G proteins (Gα, Gβ/Gγ subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane α-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Gα subunit. This leads to the dissociation of Gβ/Gγ dimer from Gα. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Gα-GTP is hydrolyzed to GDP and Gα becomes inactive (Gα-GDP), which leads to its re-association with the Gβ/Gγ dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.Key words: heterotrimeric G proteins, GPCRs, seven-transmembrane receptors, signal transduction, stress signaling     

Gastrin-stimulated Gα13 Activation of Rgnef Protein (ArhGEF28) in DLD-1 Colon Carcinoma Cells

The guanine nucleotide exchange factor Rgnef (also known as ArhGEF28 or p190RhoGEF) promotes colon carcinoma cell motility and tumor progression via interaction with focal adhesion kinase (FAK). Mechanisms of Rgnef activation downstream of integrin or G protein-coupled receptors remain undefined. In the absence of a recognized G protein signaling homology domain in Rgnef, no proximal linkage to G proteins was known. Utilizing multiple methods, we have identified Rgnef as a new effector for Gα₁₃ downstream of gastrin and the type 2 cholecystokinin receptor. In DLD-1 colon carcinoma cells depleted of Gα₁₃, gastrin-induced FAK Tyr(P)-397 and paxillin Tyr(P)-31 phosphorylation were reduced. RhoA GTP binding and promoter activity were increased by Rgnef in combination with active Gα₁₃. Rgnef co-immunoprecipitated with activated Gα₁₃Q226L but not Gα₁₂Q229L. The Rgnef C-terminal (CT, 1279–1582) region was sufficient for co-immunoprecipitation, and Rgnef-CT exogenous expression prevented Gα₁₃-stimulated SRE activity. A domain at the C terminus of the protein close to the FAK binding domain is necessary to bind to Gα₁₃. Point mutations of Rgnef-CT residues disrupt association with active Gα₁₃ but not Gα_q. These results show that Rgnef functions as an effector of Gα₁₃ signaling and that this linkage may mediate FAK activation in DLD-1 colon carcinoma cells.     

Na/H Exchanger Regulatory Factors Control Parathyroid Hormone Receptor Signaling by Facilitating Differential Activation of Gα Protein Subunits

The Na/H exchanger regulatory factors, NHERF1 and NHERF2, are adapter proteins involved in targeting and assembly of protein complexes. The parathyroid hormone receptor (PTHR) interacts with both NHERF1 and NHERF2. The NHERF proteins toggle PTHR signaling from predominantly activation of adenylyl cyclase in the absence of NHERF to principally stimulation of phospholipase C when the NHERF proteins are expressed. We hypothesized that this signaling switch occurs at the level of the G protein. We measured G protein activation by [³⁵S]GTPγS binding and Gα subtype-specific immunoprecipitation using three different cellular models of PTHR signaling. These studies revealed that PTHR interactions with NHERF1 enhance receptor-mediated stimulation of Gα_q but have no effect on stimulation of Gα_i or Gα_s. In contrast, PTHR associations with NHERF2 enhance receptor-mediated stimulation of both Gα_q and Gα_i but decrease stimulation of Gα_s. Consistent with these functional data, NHERF2 formed cellular complexes with both Gα_q and Gα_i, whereas NHERF1 was found to interact only with Gα_q. These findings demonstrate that NHERF interactions regulate PTHR signaling at the level of G proteins and that NHERF1 and NHERF2 exhibit isotype-specific effects on G protein activation.     

Functional Comparison of Rod and Cone Gαt on the Regulation of Light Sensitivity

The signaling cascades mediated by G protein-coupled receptors (GPCRs) exhibit a wide spectrum of spatial and temporal response properties to fulfill diverse physiological demands. However, the mechanisms that shape the signaling response of the GPCR are not well understood. In this study, we replaced cone transducin α (cTα) for rod transducin α (rTα) in rod photoreceptors of transgenic mice, which also express S opsin, to evaluate the role of Gα subtype on signal amplification from different GPCRs in the same cell; such analysis may explain functional differences between retinal rod and cone photoreceptors. We showed that ectopically expressed cTα 1) forms a heterotrimeric complex with rod Gβ₁γ₁, 2) substitutes equally for rTα in generating photoresponses initiated by either rhodopsin or S-cone opsin, and 3) exhibited similar light-activated translocation as endogenous rTα in rods and endogenous cTα in cones. Thus, rTα and cTα appear functionally interchangeable. Interestingly, light sensitivity appeared to correlate with the concentration of cTα when expression is reduced below 35% of normal. However, quantification of endogenous cTα concentration in cones showed a higher level to rTα in rods. Thus, reduced sensitivity in cones cannot be explained by reduced coupling efficiency between the GPCR and G protein or a lower concentration of G protein in cones versus rods.     

Macromolecular Composition Dictates Receptor and G Protein Selectivity of Regulator of G Protein Signaling (RGS) 7 and 9-2 Protein Complexes in Living Cells

Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by μ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein β subunit Gβ5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.     

Drosophila GoLoco-Protein Pins Is a Target of Gαo-mediated G Protein–coupled Receptor Signaling

G protein–coupled receptors (GPCRs) transduce their signals through trimeric G proteins, inducing guanine nucleotide exchange on their Gα-subunits; the resulting Gα-GTP transmits the signal further inside the cell. GoLoco domains present in many proteins play important roles in multiple trimeric G protein–dependent activities, physically binding Gα-subunits of the Gα_i/o class. In most cases GoLoco binds exclusively to the GDP-loaded form of the Gα-subunits. Here we demonstrate that the poly-GoLoco–containing protein Pins of Drosophila can bind to both GDP- and GTP-forms of Drosophila Gα_o. We identify Pins GoLoco domain 1 as necessary and sufficient for this unusual interaction with Gα_o-GTP. We further pinpoint a lysine residue located centrally in this domain as necessary for the interaction. Our studies thus identify Drosophila Pins as a target of Gα_o-mediated GPCR receptor signaling, e.g., in the context of the nervous system development, where Gα_o acts downstream from Frizzled and redundantly with Gα_i to control the asymmetry of cell divisions.     

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP₃)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP₃ and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP₃/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP₃ and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.     

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gα_i/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gα_i1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gα_i1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gα_o-AlF₄⁻. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gα_i1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gα_o-AlF₄⁻ and an AlF₄⁻-insensitive mutant (G42R) of Gα_i1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gα_i1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gα_o-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.     

Pivotal Role of Extended Linker 2 in the Activation of Gα by G Protein-coupled Receptor

G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gα_s mutant proteins (from a screen of 66 Gα_s mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gα_s. This extended Linker 2 is the target site of a natural product inhibitor of G_q. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β₂/β₃ loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation.     

Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

The α_1A-AR is thought to couple predominantly to the Gα_q/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gα_q coupling-defective variants of α_1A-AR, as well as a combination of Ca²⁺ channel blockers, uncovered cross-talk between α_1A-AR and β₂-AR that leads to potentiation of a Gα_q-independent signaling cascade in response to α_1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α_1A-AR. α_1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α_1A-AR. Iso induced signaling at α_1A-AR was further interrogated by probing steps along the Gα_q /PLC, Gα_s and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α_1A-AR, and CHO_α_1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca²⁺ mobilization that could be blocked by α_1A-AR selective antagonists. The kinetics of Iso induced Ca²⁺ transients differed from typical Gα_q- mediated Ca²⁺ mobilization, lacking both the fast IP₃R mediated response and the sustained phase of Ca²⁺ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α_1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gα_q.     

GRK2 Targeted Knock-down Results in Spontaneous Hypertension,and Altered Vascular GPCR Signaling

Hypertension, elevated arterial pressure, occurs as the consequence of increased peripheral resistance. G protein-coupled receptors (GPCRs) contribute to the regulation of vasodilator and vasoconstrictor responses, and their activity is regulated by a family of GPCR kinases (GRKs). GRK2 expression is increased in hypertension and this facilitates the development of the hypertensive state by increasing the desensitization of GPCRs important for vasodilation. We demonstrate here, that genetic knockdown of GRK2 using a small hairpin (sh) RNA results in altered vascular reactivity and the development of hypertension between 8–12 weeks of age in shGRK2 mice due to enhanced Gα_q/11 signaling. Vascular smooth muscle cells (VSMCs) cultured from shGRK2 knockdown mice show increases in GPCR-mediated Gα_s and Gα_q/11 signaling, as the consequence of reduced GRK2-mediated desensitization. In addition, agonists and biased agonists exhibited age-dependent alterations in ERK1/2 and Akt signaling, as well as cell proliferation and migration responses in shGRK2 knockdown VSMCs when cultured from mice that are either 3 months or 6 months of age. Changes in angiotensin II-stimulated ERK1/2 phosphorylation are observed in VSMCs derived from 6-week-old shGRK2 mice prior to the development of the hypertensive phenotype. Thus, our findings indicate that the balance between mechanisms regulating vascular tone are shifted to favor vasoconstriction in the absence of GRK2 expression and that this leads to the age-dependent development of hypertension, as a consequence of global alterations in GPCR signaling. Consequently, therapeutic strategies that target GRK2 activity, not expression, may be more effective for the treatment of hypertension.