Epitope-tagged Gq alpha subunits: expression of GTPase-deficient alpha subunits persistently stimulates phosphatidylinositol-specific phospholipase C but not mitogen-activated protein kinase activity regulated by the M1 muscarinic acetylcholine receptor.

The regulation of gene expression by cell surface receptors often involves the stimulation of signaling pathways including one or more members of the MAPK superfamily of serine-threonine kinases. Upon their activation in the cytosol, MAPKs can translocate to the nucleus and affect the activity of a variety of transcription factors. Recently, it has been observed that a novel member of the MAPK superfamily, ERK5, can be potently activated by transforming G protein-coupled receptors (GPCRs) and that ERK5 participates in the regulation of c-jun expression through the activation of MEF2 transcription factors. How cell surface receptors, including GPCRs, stimulate ERK5 is still poorly understood. In this study, we have used transiently transfected COS-7 cells to begin delineating the biochemical route linking GPCRs to ERK5. We show that receptors that can couple to the G q and G 12/13 families of heterotrimeric G proteins, m1 and thrombin receptors, respectively, but not those coupled to G i , such as m2 receptors, are able to regulate the activity of ERK5. To investigate which heterotrimeric G proteins signal to ERK5, we used a chimeric system by which G␣ q -and G␣ 13 -mediated signaling pathways can be conditionally activated upon ligand stimulation. Using this system, as well as the expression of activated forms of G protein subunits, we show that the G␣ q and G␣ 12/13 families of heterotrimeric G proteins, but not the G␣ i , G␣ s , and ␤␥ subunits, are able to regulate ERK5. Furthermore, we provide evidence that the stimulation of ERK5 by GPCRs involves a novel signaling pathway, which is distinct from those regulated by Ras and Rho GTPases.

Section: Resultsmentioning

confidence: 99%

Signaling from G Protein-coupled Receptors to ERK5/Big MAPK 1 Involves Gαq and Gα12/13 Families of Heterotrimeric G Proteins

Fukuhara

Chiariello

et al. 2000

“…To our knowledge, all GAP-deficient RGS mutants described so far have significantly reduced G␣ binding affinity (24 -26), which in turn reduces not only their GAP activity but also potential effector antagonistic effects. Therefore, we chose to take advantage of a well characterized point mutation in G␣ q * (G␣ q Q209L) that destroys its GTPase activity and thereby renders it constitutively active (12). Both PLC␤ and RGS proteins bind to receptor-activated as well as GTPase-deficient G␣ subunits.…”

Section: Discussionmentioning

confidence: 99%

“…M 3 receptors were expected to couple to endogenously expressed G q , which is subject to the activation/inactivation cycle characteristic for heterotrimeric G proteins. In contrast, G␣ q * is inert to GTP hydrolysis and therefore does not depend on receptor activation to stimulate PLC␤ (12). Comparing the inhibitory effect of each RGS protein in both settings provided direct insight about the functional importance of GTPase activation for the inhibitory effect of RGS proteins on G q -mediated signaling in vivo.…”

mentioning

confidence: 99%

Differential Contribution of GTPase Activation and Effector Antagonism to the Inhibitory Effect of RGS Proteins on Gq-mediated Signaling in Vivo

Anger

Zhang

Mende

2004

RGS proteins act as negative regulators of G protein signaling by serving as GTPase-activating proteins (GAP) for ␣ subunits of heterotrimeric G proteins (G␣), thereby accelerating G protein inactivation. RGS proteins can also block G␣-mediated signal production by competing with downstream effectors for G␣ binding. Little is known about the relative contribution of GAP and effector antagonism to the inhibitory effect of RGS proteins on G protein-mediated signaling. By comparing the inhibitory effect of RGS2, RGS3, RGS5, and RGS16 on G␣ q -mediated phospholipase C␤ (PLC␤) activation under conditions where GTPase activation is possible versus nonexistent, we demonstrate that members of the R4 RGS subfamily differ significantly in their dependence on GTPase acceleration. COS-7 cells were transiently transfected with either muscarinic M 3 receptors, which couple to endogenous G q protein and mediate a stimulatory effect of carbachol on PLC␤, or constitutively active G␣ q * , which is inert to GTP hydrolysis and activates PLC␤ independent of receptor activation. In M 3 -expressing cells, all of the RGS proteins significantly blunted the efficacy and potency of carbachol. In contrast, G␣ q * -induced PLC␤ activation was inhibited by RGS2 and RGS3 but not RGS5 and RGS16. The observed differential effects were not due to changes in M 3 , G␣ q / G␣ q * , PLC␤, or RGS expression, as shown by receptor binding assays and Western blots. We conclude that closely related R4 RGS family members differ in their mechanism of action. RGS5 and RGS16 appear to depend on G protein inactivation, whereas GAP-independent mechanisms (such as effector antagonism) are sufficient to mediate the inhibitory effect of RGS2 and RGS3.Many extracellular stimuli elicit intracellular responses by activating seven-transmembrane receptors that are coupled to heterotrimeric G proteins comprising ␣ and ␤␥ subunits (1, 2). The duration of the response of a cell to external signals is largely determined by the activation/inactivation cycle of G proteins. Activated receptors trigger GTP-for-GDP exchange on G␣ subunits, thus dissociating G␣ from G␤ ␥ and subsequent activation of downstream effectors (such as enzymes and ion channels). The duration of G protein activation is limited by GTPase activity intrinsic to G␣ subunits that catalyzes the conversion of active GTP-bound G␣ into inactive GDP-bound G␣, which in turn can reassociate with G␤␥ and receptors.RGS proteins belong to a family of more than 20 proteins with a conserved RGS core domain of ϳ120 amino acids that is necessary and sufficient for binding to G␣ subunits (3). They are divided into several subfamilies based on their structural similarities, gene organization, and function. RGS proteins act as regulators of G protein signaling by limiting the signals generated by G protein-coupled receptors. They markedly increase the rate at which G␣ subunits hydrolyze GTP to GDP, a property that defines them as GTPase-activating proteins (or GAPs) 1 (4). Hastening of G␣ inactivation facilitates the reass...

“…Since the TSH-induced activation of PLC is mediated by the TSHR activation of G q /G 11 (10), we planned to provide activated G q /G 11 to the cells to directly activate PLC without TSH. For this purpose, the cells were transfected with a constitutively active G 11 ␣ mutant, G 11 ␣(Q209L), which lacks GTPase activity (44) (Fig. 6), or challenged with NaF, a nonselective G protein activator (Fig.…”

Section: A Single Type Of Ptx-sensitive G Protein G I 2 or G I 3 Mementioning

confidence: 99%

βγ Subunits of Pertussis Toxin-sensitive G Proteins Mediate A1 Adenosine Receptor Agonist-induced Activation of Phospholipase C in Collaboration with Thyrotropin

Tomura

Itoh

Sho

et al. 1997

COS-7 cells were transiently transfected with human thyrotropin receptor and dog A 1 adenosine receptor cDNAs. An A 1 agonist, N 6 -(L-2-phenylisopropyl) adenosine (PIA), which is ineffective alone, enhanced the thyrotropin (TSH)-induced inositol phosphate production, reflecting phospholipase C (PLC) activation, but inhibited the TSH-induced cAMP accumulation, reflecting adenylyl cyclase inhibition. These PIA-induced actions were completely inhibited by pertussis toxin (PTX) treatment. Moreover, in the cells expressing a PTX-insensitive mutant of G i 2␣ or G i 3␣, in which a glycine residue was substituted for a cysteine residue to be ADP-ribosylated by PTX, at the fourth position of the C terminus, PIA effectively exerted both stimulatory and inhibitory effects on the TSH-induced actions although the cells were treated with the toxin. Overexpression of the ␤␥ subunits of the G proteins enhanced the TSHinduced inositol phosphate production without any significant effect on the cAMP response; under these conditions, PIA did not further increase the elevated inositol phosphate response to TSH. On the contrary, overexpression of a constitutively active mutant of G i 2␣, in which the guanosine triphosphatase activity is lost, inhibited the TSH-induced cAMP accumulation but hardly affected the inositol phosphate response; under these conditions, PIA never exerted further inhibitory effects on the cAMP response to TSH. In contrast to the case of the TSH-induced inositol phosphate response, the response to a constitutively active G 11 ␣ mutant was not appreciably affected, and that to NaF was rather inhibited by PIA and overexpression of the ␤␥ subunits. Taken together, these results suggest that a single type of PTX-sensitive G protein mediates the A 1 adenosine receptor-linked modulation of two signaling pathways in collaboration with an activated thyrotropin receptor; ␣ subunits of the PTX-sensitive G proteins mediate the inhibitory action on adenylyl cyclase, and the ␤␥ subunits mediate the stimulatory action on PLC. In the case of the latter stimulatory action on PLC, the ␤␥ subunits may not directly activate PLC. The possible mechanism by which ␤␥ subunits enhance the TSH-induced PLC activation is discussed.