EDITORIAL FOCUS
Focus on "Targeted expression of activated Q227L Galpha s in vivo"

Susan F. Steinberg

Departments of Pharmacology and Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032


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GS was identified over 20 years ago as a heterotrimeric protein required for hormone-dependent activation of adenylyl cyclase in liver membranes (10). Since those original studies, Gs has come to be recognized as a ubiquitously expressed G protein family member that relays signals from a diverse array of heptahelical receptors to adenylyl cyclase as well as other effectors. Gs is composed of an alpha s-subunit (which contains the guanine nucleotide binding site) complexed with a tightly associated dimer of beta - and gamma -subunits (2). The basic model of G protein activation involves two independent cycles, with both guanine nucleotide exchange and subunit dissociation regulating Gs activity. In the basal state, Gs exists as an inactive GDP-liganded alpha s-beta gamma heterotrimer. G protein activation promotes a conformational change that catalyzes the exchange of GDP for GTP on the alpha s-subunit. This results in the dissociation of alpha s from beta gamma -dimers, allowing both the freed GTP-liganded alpha s-subunit and the liberated beta gamma -dimers to regulate effectors. Signal termination is via hydrolysis of GTP to GDP by the intrinsic GTPase of alpha s. The GDP-liganded alpha s reassociates with beta gamma -dimers to reform inactive heterotrimers (thereby preventing further beta gamma -dimer modulation of effectors). However, recent studies identify RGS-PX1 as a bifunctional protein that acts both as a "regulator of G protein signaling" (RGS) to accelerate GTP hydrolysis on Gs and a regulator of endocytic trafficking to lysosomes. The link between G protein signaling and membrane trafficking events may be a common theme that introduces spatial and temporal constraints on signaling protein interactions in native cells (15).

Several clinical disorders have been linked to aberrant Gs signaling. The pathogenic exotoxins of Vibrio cholerae and exotoxins of certain strains of Escherichia coli catalyze the ADP-ribosylation of a key arginine residue at position 201 in the GTPase domain of alpha s. Mutagenesis and X-ray crystallographic studies identify this residue as critical for the proper orientation of the gamma -phosphate of GTP and efficient GTP hydrolysis (12). Covalent modification of Arg201 slows the inherent GTPase activity of alpha s and impairs signal termination. This results in a constitutively activated alpha s, which persistently stimulates adenylyl cyclase (even in the absence of ligand). The rise in cAMP levels in intestinal mucosal cells promotes salt and water secretion, leading to profuse watery diarrhea.

Mutations that inhibit the intrinsic GTPase activity of alpha s also have been identified in many endocrine tumors, where cAMP is a mitogenic signal (13). Oncogenic Galpha s mutations have been mapped to Arg201 (the site for ADP-ribosylation by cholera toxin) as well as an adjacent glutamine (which also contributes to optimal GTP gamma -phosphate alignment and efficient GTP hydrolysis). The Arg201-activating alpha s mutation has also been identified in affected tissues of patients with McCune-Albright syndrome (MAS; Ref. 7). This is a sporadic congenital disorder caused by a dominant somatic mutation in early development; the clinical features of MAS are dictated by the distribution of cells bearing the mutation. MAS generally is characterized by the triad of scattered areas of hyperpigmentation (café au lait spots), polyostotic fibrous dysplasia, and autonomous hyperfunction of one or more endocrine glands (leading to precocious puberty, hyperthyroidism, Cushing syndrome, or acromegaly). When MAS abnormalities are restricted to bone, skin, and endocrine organs, there is little effect on mortality. However, some patients with MAS manifest a more severe form of the disorder, with more widespread abnormalities beyond the traditional target tissues. This presumably reflects a more widespread distribution of the Galpha s mutation. For example, the constellation of cardiac abnormalities identified in some patients with MAS (cardiomegaly, arrhythmias, and sudden death in infancy) is consistent with elevated Galpha s activity in myocardial tissue.

Transgenic mice that overexpress normal or mutationally activated Galpha s in specific tissues provide models to explore the functional role of Galpha s in human disease. Cardiac-selective overexpression of wild-type Galpha s has been accomplished with the alpha -myosin heavy chain (alpha -MHC) promoter. This model was conceived as a paradigm to decipher the consequences of chronic catecholamine stimulation of Gs-coupled beta -adrenergic receptors in heart failure. Transgenic mice with a three- to fourfold increase in Galpha s protein expression (and approximately a doubling in Galpha s activity) display augmented inotropic and chronotropic responses to sympathomimetic amines with normal cardiac architecture at 10 mo of age. However, this phenotype gives way to a dilated cardiomyopathy, with reduced left ventricular ejection fraction, interstitial fibrosis, cellular hypertrophy and apoptosis, ventricular arrhythmias, and sudden death as the mice age (6). These studies argue that Galpha s plays a complex role, both to provide critical inotropic support and to promote tissue remodeling during the evolution of heart failure.

In the current article in focus (Ref. 5; see p. C386 in this issue) Huang et al. have used a similar molecular approach to target the constitutively activated Q227L Galpha s mutant to fat, liver, and skeletal muscle tissues [using the phosphoenolpyruvate carboxykinase (PEPCK) promoter]. Like the alpha -MHC promoter, the PEPCK promoter drives expression only in postnatal tissues, obviating the concern that Galpha s overexpression in utero would reduce fetal viability. Biochemical characterization of this mouse reveals a 40-50% increase in Galpha s protein expression in targeted tissues, a surprisingly modest increase in basal (but not agonist or forskolin activated) cAMP levels and no obvious gross macroscopic phenotype. The very modest changes in cAMP in affected tissues led these authors to consider compensatory mechanisms that might mitigate cAMP accumulation during unabated Q227L Galpha s stimulation. Indeed, modest increases in Galpha i2 and cAMP-specific phosphodiesterase, which would decrease cAMP accumulation, were detected in affected tissues. Reduced in vitro measurements of cAMP-regulated protein kinase (PKA) activity were also detected in the affected tissues and were interpreted as a mechanism to diminish cAMP actions. However, immunoblot analyses of PKA regulatory and catalytic subunit expression identified increases in PKA regulatory subunit expression with no change in PKA catalytic subunit expression. These types of changes in PKA protein subunit expression actually might serve to reroute (rather than reduce) PKA phosphorylation pathways in intact cells, and they deserve further study in more physiological models. To the extent that changes in G proteins, phosphodiesterases, and PKA mimic adaptive responses observed in other conditions associated with increased cAMP signaling (MAS, heart failure), the changes identified in this model might represent a compensatory response to chronically enhanced cAMP signaling in the affected tissue. However, the Q227L Galpha s transgenic mice display delayed rectification of blood glucose during glucose challenge. To the extent that Q227L Galpha s overexpression in metabolically active tissues (such as liver, adipose, and skeletal muscle) leads to a generalized metabolic disorder, alternative mechanism(s) whereby Q227L Galpha s overexpression can induce generalized changes in signaling protein expression/action in both affected and bystander tissues also must be considered in future studies.

Like most molecular models, transgenic mice with PEPCK-driven Q227L Galpha s expression are likely to be best suited to investigate certain aspects of Galpha s signaling. For example, the molecular model developed by Huang et al. (5) uses a Q227L Galpha s mutant identified in certain endocrine tumors and a promoter that drives expression in adipose, liver, and skeletal muscle. Although neither the mutant construct nor the tissue distribution mimics the abnormalities identified in MAS, this model is likely to provide a generally useful strategy to explore the consequences of Galpha s activation, particularly in the context of disorders associated with constitutively activated mutant proteins (endocrine adenomas, MAS) or in disorders affecting metabolically active tissues. Other promoters that selectively drive expression in other tissues might induce a very different phenotype and identify tissue-specific differences in the consequences of Gs activation. However, to the extent that physiological Galpha s interactions with beta gamma -dimers critically influence the fidelity and specificity of heterotrimeric G protein interactions with G protein-coupled receptors and effectors, models of Q227L Galpha s overexpression (which might bind but not be regulated in a traditional manner by RGS proteins and beta gamma -dimers) eventually are likely to prove inadequate for studies of the intricacies of physiological Gs signaling. The very nature of the results obtained in molecular models of Galpha s overexpression, where substantial increases in Galpha s expression lead to only relatively modest increases in cAMP levels, suggests that the stoichiometry of individual elements in the signaling cascade (receptor, G protein, effector) and potential compartmentation of signaling pathways also critically influence functional phenotypes and deserve attention.

Huang et al. (5) have developed a model that should reveal clinically important Galpha s effectors. Although Galpha s-dependent activation of adenylyl cyclase and enhanced signaling via the traditional cAMP/PKA pathway largely can explain the contractile phenotype induced by cardiac Galpha s overexpression, the mechanism(s) whereby increased Galpha s signaling leads to cellular remodeling and changes in the regulation of growth is less straightforward. p38-mitogen-activated protein kinase (MAPK) has been identified downstream from PKA in cardiomyocytes, where it provides a potential mechanism to explain the more chronic deleterious changes induced by Galpha s overexpression in the heart (16). In other tissues, Galpha s/cAMP modulation of growth results from complex and highly tissue-specific regulatory controls. In vascular smooth muscle cells, cAMP antagonizes growth factor-dependent activation of the Raf/extracellular signal-regulated kinase (ERK) cascade and proliferation. The inhibitory effects of cAMP are sufficiently robust in this cell type that adenovirus-mediated delivery of constitutively activated Galpha s has been evaluated as a strategy to inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia in models of vessel injury (4). Inhibitory modulation of ERK by cAMP has been attributed to PKA-dependent phosphorylation of c-Raf (at Ser43, Ser259, and Ser259), which inhibits signaling via the MAPK kinase (MEK)/ERK cascade at least in part by preventing c-Raf-Ras interactions (1, 14). Constitutively activated Galpha s (and elevated cAMP) activates ERK and stimulates proliferation in cell types (including endocrine tumors) that express B-Raf, a different Raf isoform that lacks a PKA site corresponding to Ser43 and displays a more restricted tissue distribution. Here, the effects of cAMP/PKA to activate ERK and stimulate proliferation have been attributed to a parallel pathway involving Rap-1 (a member of the Ras superfamily of small GTP-binding proteins that shares structural homology with Ras in its effector domain) and B-Raf. Both PKA-dependent pathway and PKA-independent/cAMP-dependent mechanisms (involving the cAMP-activated guanine nucleotide exchange factor, Epac I) promote GTP loading of Rap-1 and activation of the B-Raf/ERK phosphorylation cascade (8, 11). Finally, recent studies identify Src family tyrosine kinases as additional nontraditional alternative targets of Galpha s that could initiate signaling via the ERK cascade as well as other pathways (3, 9).

Direct consequences of Q227L Galpha s transgene overexpression are not always easily distinguished from secondary compensatory changes that develop over the life of the animal (and with the development of a diseased phenotype). Nevertheless, the mouse model developed by Huang et al. (5) is well suited for studies that delve into mechanisms activated by Gs and their role in biochemical/structural remodeling and growth control. The more formidable challenge of future studies will be to integrate concepts of signaling protein stoichiometry, targeting, and protein-protein interactions into this and other models of Galpha s function, to fully understand the exquisite (and seemingly contradictory) high level of specificity and interdependence on other signaling molecules achieved by the Galpha s protein.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-28958.


    FOOTNOTES

Address for reprint requests and other correspondence: S. F. Steinberg, Dept. of Pharmacology, College of Physicians and Surgeons, Columbia Univ., 630 West 168 St., New York, NY 10032 (E-mail: sfs1{at}columbia.edu).

10.1152/ajpcell.00198.2002


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Am J Physiol Cell Physiol 283(2):C383-C385
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society




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