G0/G1 Switch Gene 2 Regulates Cardiac Lipolysis

The anabolism and catabolism of myocardial triacylglycerol (TAG) stores are important processes for normal cardiac function. TAG synthesis detoxifies and stockpiles fatty acids to prevent lipotoxicity, whereas TAG hydrolysis (lipolysis) remobilizes fatty acids from endogenous storage pools as energy substrates, signaling molecules, or precursors for complex lipids. This study focused on the role of G₀/G₁ switch 2 (G0S2) protein, which was previously shown to inhibit the principal TAG hydrolase adipose triglyceride lipase (ATGL), in the regulation of cardiac lipolysis. Using wild-type and mutant mice, we show the following: (i) G0S2 is expressed in the heart and regulated by the nutritional status with highest expression levels after re-feeding. (ii) Cardiac-specific overexpression of G0S2 inhibits cardiac lipolysis by direct protein-protein interaction with ATGL. This leads to severe cardiac steatosis. The steatotic hearts caused by G0S2 overexpression are less prone to fibrotic remodeling or cardiac dysfunction than hearts with a lipolytic defect due to ATGL deficiency. (iii) Conversely to the phenotype of transgenic mice, G0S2 deficiency results in a de-repression of cardiac lipolysis and decreased cardiac TAG content. We conclude that G0S2 acts as a potent ATGL inhibitor in the heart modulating cardiac substrate utilization by regulating cardiac lipolysis.     

Defective Adipose Lipolysis and Altered Global Energy Metabolism in Mice with Adipose Overexpression of the Lipolytic Inhibitor G0/G1 Switch Gene 2 (G0S2)

Biochemical and cell-based studies have identified the G0S2 (G₀/G₁ switch gene 2) as a selective inhibitor of the key intracellular triacylglycerol hydrolase, adipose triglyceride lipase. To better understand the physiological role of G0S2, we constructed an adipose tissue-specific G0S2 transgenic mouse model. In comparison with wild type animals, the transgenic mice exhibited a significant increase in overall fat mass and a decrease in peripheral triglyceride accumulation. Basal and adrenergically stimulated lipolysis was attenuated in adipose explants isolated from the transgenic mice. Following fasting or injection of a β3-adrenergic agonist, in vivo lipolysis and ketogenesis were decreased in G0S2 transgenic mice when compared with wild type animals. Consequently, adipose overexpression of G0S2 prevented the “switch” of energy substrate from carbohydrates to fatty acids during fasting. Moreover, G0S2 overexpression promoted accumulation of more and larger lipid droplets in brown adipocytes without impacting either mitochondrial morphology or expression of oxidative genes. This phenotypic change was accompanied by defective cold adaptation. Furthermore, feeding with a high fat diet caused a greater gain of both body weight and adiposity in the transgenic mice. The transgenic mice also displayed a decrease in fasting plasma levels of free fatty acid, triglyceride, and insulin as well as improved glucose and insulin tolerance. Cumulatively, these results indicate that fat-specific G0S2 overexpression uncouples adiposity from insulin sensitivity and overall metabolic health through inhibiting adipose lipolysis and decreasing circulating fatty acids.     

G0/G1 switch gene-2 regulates human adipocyte lipolysis by affecting activity and localization of adipose triglyceride lipase

The hydrolysis of triglycerides in adipocytes, termed lipolysis, provides free fatty acids as energy fuel. Murine lipolysis largely depends on the activity of adipose triglyceride lipase (ATGL), which is regulated by two proteins annotated as comparative gene identification-58 (CGI-58) and G0/G1 switch gene-2 (G0S2). CGI-58 activates and G0S2 inhibits ATGL activity. In contrast to mice, the functional role of G0S2 in human adipocyte lipolysis is poorly characterized. Here we show that overexpression or silencing of G0S2 in human SGBS adipocytes decreases and increases lipolysis, respectively. Human G0S2 is upregulated during adipocyte differentiation and inhibits ATGL activity in a dose-dependent manner. Interestingly, C-terminally truncated ATGL mutants, which fail to localize to lipid droplets, translocate to the lipid droplet upon coexpression with G0S2, suggesting that G0S2 anchors ATGL to lipid droplets independent of ATGL''s C-terminal lipid binding domain. Taken together, our results indicate that G0S2 also regulates human lipolysis by affecting enzyme activity and intracellular localization of ATGL. Increased lipolysis is known to contribute to the pathogenesis of insulin resistance, and G0S2 expression has been shown to be reduced in poorly controlled type 2 diabetic patients. Our data indicate that downregulation of G0S2 in adipose tissue could represent one of the underlying causes leading to increased lipolysis in the insulin-resistant state.     

Relative contribution of adipose triglyceride lipase and hormone-sensitive lipase to tumor necrosis factor-α (TNF-α)-induced lipolysis in adipocytes

TNF-α potently stimulates basal lipolysis in adipocytes, which may contribute to hyperlipidemia and peripheral insulin resistance in obesity. Recent studies show that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) act sequentially in catalyzing the first two steps of adipose lipolysis in response to β-adrenergic stimulation. Here, we sought to determine their functional roles in TNF-α-induced lipolysis. Silencing of ATGL expression in adipocytes almost completely abolished basal and TNF-α-induced glycerol release. In comparison, the glycerol release under the same conditions was only partially decreased upon reduction in expression of either HSL or the ATGL coactivator CGI-58. Interestingly, overexpression of ATGL restored the lipolytic rates in cells with silenced HSL or CGI-58, indicating a predominant role for ATGL. While expression of ATGL, HSL and CGI-58 remains mostly unaffected, TNF-α treatment caused a rapid abrogation of the ATGL inhibitory protein G0S2. TNF-α drastically decreased the level of G0S2 mRNA, and the level of G0S2 protein could be maintained by inhibiting proteasomal protein degradation using MG-132. Furthermore, coexpression of G0S2 was able to significantly decrease TNF-α-stimulated lipolysis mediated by overexpressed ATGL or CGI-58. We propose that the early reduction in G0S2 content is permissive for TNF-α-induced lipolysis.     

Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA–independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.     

Characterization of Lipolytic Inhibitor G(0)/G(1) Switch Gene-2 Protein (G0S2) Expression in Male Sprague-Dawley Rat Skeletal Muscle Compared to Relative Content of Adipose Triglyceride Lipase (ATGL) and Comparitive Gene Identification-58 (CGI-58)

The rate-limiting enzyme in lipolysis, adipose triglyceride lipase (ATGL), is activated by comparative gene identification-58 (CGI-58) and inhibited by the G(0)/G(1) switch gene-2 (G0S2) protein. It is speculated that inhibition of ATGL is through a dose dependent manner of relative G0S2 protein content. There is little work examining G0S2 expression in lipolytic tissues, and the relative expression across oxidative tissues such as skeletal muscle has not yet been described. Three muscles, soleus (SOL), red gastrocnemius (RG), and white gastrocnemius (WG) were excised from 57-day old male Sprague-Dawley rats (n = 9). QRT-PCR was used for mRNA analysis, and western blotting was conducted to determine protein content. ATGL and G0S2 protein content were both greatest in the lipolytic SOL, with the least amount of both ATGL and G0S2 protein content found in the WG. CGI-58 protein content however did not mirror ATGL and G0S2 protein content, since the RG had the greatest CGI-58 protein content when compared to the SOL and WG. When comparing our tissues based on CGI-58-to-ATGL ratio and G0S2-to-ATGL ratio, it was discovered that contrary to oxidative demand, the glycolytic WG had the greatest activator CGI-58-to-ATGL ratio with the oxidative SOL having the least, and no differences in G0S2-to-ATGL across the three muscle types. These data suggest that the content of G0S2 relative to the lipase in skeletal muscle would not predict lipolytic potential.     

Residues of the minimal sequence of G0S2 collectively contribute to ATGL inhibition while C-and N-terminal extensions promote binding to ATGL

The protein encoded by the G0/G1 switch gene 2 (G0S2) is a potent inhibitor of adipose triglyceride lipase (ATGL) and thus an important regulator of intracellular lipolysis. Since dysfunction of lipolysis is associated with metabolic diseases including diabetes and obesity, inhibition of ATGL is considered a therapeutic strategy. G0S2 interacts with ATGL's patatin-domain to mediate non-competitive inhibition, however atomic details of the inhibition mechanism are incompletely understood. Sequences of G0S2 from higher organisms show a highly conserved N-terminal part, including a hydrophobic region covering amino acids 27 to 42. We show that predicted G0S2 orthologs from platypus, chicken and Japanese rice-fish are able to inhibit human and mouse ATGL, emphasizing the contribution of conserved amino acid to ATGL inhibition. Our site directed mutagenesis and truncation studies give insights in the protein-protein interaction on a per-residue level. We determine that the minimal sequence required for ATGL inhibition ranges from amino acids 20 to 44. Residues Y27, V28, G30, A34 G37, V39 or L42 within this sequence play a substantial role in ATGL inhibition. Furthermore, we show that unspecific interactions of the N-terminal part (amino acids 20-27) of the minimal sequence facilitate the interaction to ATGL. Our studies also demonstrate that full-length G0S2 shows higher tolerance to specific single amino acid exchanges in the hydrophobic region due to the stronger contributions of unspecific interactions. However, exchanges of more than one amino-acid in the hydrophobic region also result in the loss of function as ATGL inhibitor even in the full-length protein.     

The G0/G1 switch gene 2 (G0S2): Regulating metabolism and beyond

The G0/G1 switch gene 2 (G0S2) was originally identified in blood mononuclear cells following induced cell cycle progression. Translation of G0S2 results in a small basic protein of 103 amino acids in size. It was initially believed that G0S2 mediates re-entry of cells from the G0 to G1 phase of the cell cycle. Recent studies have begun to reveal the functional aspects of G0S2 and its protein product in various cellular settings. To date the best-known function of G0S2 is its direct inhibitory capacity on the rate-limiting lipolytic enzyme adipose triglyceride lipase (ATGL). Other studies have illustrated key features of G0S2 including sub-cellular localization, expression profiles and regulation, and possible functions in cellular proliferation and differentiation. In this review we present the current knowledge base regarding all facets of G0S2, and pose a variety of questions and hypotheses pertaining to future research directions.     

Differential control of ATGL-mediated lipid droplet degradation by CGI-58 and G0S2

Lipid droplets (LDs) are intracellular storage sites for triacylglyerols (TAGs) and steryl esters, and play essential roles in energy metabolism and membrane biosynthesis. Adipose triglyceride lipase (ATGL) is the key enzyme for TAG hydrolysis (lipolysis) in adipocytes and LD degradation in nonadipocyte cells. Lipase activity of ATGL in vivo largely depends on its C-terminal sequence as well as coactivation by CGI-58. Here we demonstrate that the C-terminal hydrophobic domain in ATGL is required for LD targeting and CGI-58-independent LD degradation. Overexpression of wild type ATGL causes a dramatic decrease in LD size and number, whereas a mutant lacking the hydrophobic domain fails to localize to LDs and to affect their morphology. Interestingly, coexpression of CGI-58 is able to promote LD turnover mediated by this ATGL mutant. Recently we have discovered that G0S2 acts as an inhibitor of ATGL activity and ATGL-mediated lipolysis. Here we show that G0S2 binds to ATGL irrelevantly of its activity state or the presence of CGI-58. In G0S2-expressing cells, the combined expression of CGI-58 and ATGL is incapable of stimulating LD turnover. We propose that CGI-58 and G0S2 regulate ATGL via non-competing mechanisms.     

The minimal domain of adipose triglyceride lipase (ATGL) ranges until leucine 254 and can be activated and inhibited by CGI-58 and G0S2, respectively

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme of lipolysis. ATGL specifically hydrolyzes triacylglycerols (TGs), thereby generating diacylglycerols and free fatty acids. ATGL's enzymatic activity is co-activated by the protein comparative gene identification-58 (CGI-58) and inhibited by the protein G0/G1 switch gene 2 (G0S2). The enzyme is predicted to act through a catalytic dyad (Ser47, Asp166) located within the conserved patatin domain (Ile10-Leu178). Yet, neither an experimentally determined 3D structure nor a model of ATGL is currently available, which would help to understand how CGI-58 and G0S2 modulate ATGL's activity. In this study we determined the minimal active domain of ATGL. This minimal fragment of ATGL could still be activated and inhibited by CGI-58 and G0S2, respectively. Furthermore, we show that this minimal domain is sufficient for protein-protein interaction of ATGL with its regulatory proteins. Based on these data, we generated a 3D homology model for the minimal domain. It strengthens our experimental finding that amino acids between Leu178 and Leu254 are essential for the formation of a stable protein domain related to the patatin fold. Our data provide insights into the structure-function relationship of ATGL and indicate higher structural similarities in the N-terminal halves of mammalian patatin-like phospholipase domain containing proteins, (PNPLA1, -2,- 3 and -5) than originally anticipated.     

Lipolysis - a highly regulated multi-enzyme complex mediates the catabolism of cellular fat stores

Lipolysis is the biochemical pathway responsible for the catabolism of triacylglycerol (TAG) stored in cellular lipid droplets. The hydrolytic cleavage of TAG generates non-esterified fatty acids, which are subsequently used as energy substrates, essential precursors for lipid and membrane synthesis, or mediators in cell signaling processes. Consistent with its central importance in lipid and energy homeostasis, lipolysis occurs in essentially all tissues and cell types, it is most abundant, however, in white and brown adipose tissue. Over the last 5years, important enzymes and regulatory protein factors involved in lipolysis have been identified. These include an essential TAG hydrolase named adipose triglyceride lipase (ATGL) [annotated as patatin-like phospholipase domain-containing protein A2], the ATGL activator comparative gene identification-58 [annotated as α/β hydrolase containing protein 5], and the ATGL inhibitor G0/G1 switch gene 2. Together with the established hormone-sensitive lipase [annotated as lipase E] and monoglyceride lipase, these proteins constitute the basic "lipolytic machinery". Additionally, a large number of hormonal signaling pathways and lipid droplet-associated protein factors regulate substrate access and the activity of the "lipolysome". This review summarizes the current knowledge concerning the enzymes and regulatory processes governing lipolysis of fat stores in adipose and non-adipose tissues. Special emphasis will be given to ATGL, its regulation, and physiological function.     

The Hepatitis C Virus Core Protein Inhibits Adipose Triglyceride Lipase (ATGL)-mediated Lipid Mobilization and Enhances the ATGL Interaction with Comparative Gene Identification 58 (CGI-58) and Lipid Droplets

Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL^−/− mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs.     

The G0/G1 Switch Gene 2 Is an Important Regulator of Hepatic Triglyceride Metabolism

Nonalcoholic fatty liver disease is associated with obesity and insulin resistance. Factors that regulate the disposal of hepatic triglycerides contribute to the development of hepatic steatosis. G₀/G₁ switch gene 2 (G0S2) is a target of peroxisome proliferator-activated receptors and plays an important role in regulating lipolysis in adipocytes. Therefore, we investigated whether G0S2 plays a role in hepatic lipid metabolism. Adenovirus-mediated expression of G0S2 (Ad-G0S2) potently induced fatty liver in mice. The liver mass of Ad-G0S2-infected mice was markedly increased with excess triglyceride content compared to the control mice. G0S2 did not change cellular cholesterol levels in hepatocytes. G0S2 was found to be co-localized with adipose triglyceride lipase at the surface of lipid droplets. Hepatic G0S2 overexpression resulted in an increase in plasma Low-density lipoprotein (LDL)/Very-Low-density (VLDL) lipoprotein cholesterol level. Plasma High-density lipoprotein (HDL) cholesterol and ketone body levels were slightly decreased in Ad-G0S2 injected mice. G0S2 also increased the accumulation of neutral lipids in cultured HepG2 and L02 cells. However, G0S2 overexpression in the liver significantly improved glucose tolerance in mice. Livers expressing G0S2 exhibited increased 6-(N-(7-nitrobenz-2-oxa-1-3-diazol-4-yl) amino)-6-deoxyglucose uptake compared with livers transfected with control adenovirus. Taken together, our results provide evidence supporting an important role for G0S2 as a regulator of triglyceride content in the liver and suggest that G0S2 may be a molecular target for the treatment of insulin resistance and other obesity-related metabolic disorders.     

Unique regulation of adipose triglyceride lipase (ATGL) by perilipin 5, a lipid droplet-associated protein

Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, co-immunoprecipitation, [(32)P]orthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased [(32)P]orthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL.     

Human frame shift mutations affecting the carboxyl terminus of perilipin increase lipolysis by failing to sequester the adipose triglyceride lipase (ATGL) coactivator AB-hydrolase-containing 5 (ABHD5)

Perilipin (PLIN1) is a constitutive adipocyte lipid droplet coat protein. N-terminal amphipathic helices and central hydrophobic stretches are thought to anchor it on the lipid droplet, where it appears to function as a scaffold protein regulating lipase activity. We recently identified two different C-terminal PLIN1 frame shift mutations (Leu-404fs and Val-398fs) in patients with a novel subtype of partial lipodystrophy, hypertriglyceridemia, severe insulin resistance, and type 2 diabetes (Gandotra, S., Le Dour, C., Bottomley, W., Cervera, P., Giral, P., Reznik, Y., Charpentier, G., Auclair, M., Delépine, M., Barroso, I., Semple, R. K., Lathrop, M., Lascols, O., Capeau, J., O'Rahilly, S., Magré, J., Savage, D. B., and Vigouroux, C. (2011) N. Engl. J. Med. 364, 740-748.) When overexpressed in preadipocytes, both mutants fail to inhibit basal lipolysis. Here we used bimolecular fluorescence complementation assays to show that the mutants fail to bind ABHD5, permitting its constitutive coactivation of ATGL, resulting in increased basal lipolysis. siRNA-mediated knockdown of either ABHD5 or ATGL expression in the stably transfected cells expressing mutant PLIN1 reduced basal lipolysis. These insights from naturally occurring human variants suggest that the C terminus sequesters ABHD5 and thus inhibits basal ATGL activity. The data also suggest that pharmacological inhibition of ATGL could have therapeutic potential in patients with this rare but metabolically serious disorder.     

Adipose triglyceride lipase activity is inhibited by long-chain acyl-coenzyme A

Adipose triglyceride lipase (ATGL) is required for efficient mobilization of triglyceride (TG) stores in adipose tissue and non-adipose tissues. Therefore, ATGL strongly determines the availability of fatty acids for metabolic reactions. ATGL activity is regulated by a complex network of lipolytic and anti-lipolytic hormones. These signals control enzyme expression and the interaction of ATGL with the regulatory proteins CGI-58 and G0S2. Up to date, it was unknown whether ATGL activity is also controlled by lipid intermediates generated during lipolysis. Here we show that ATGL activity is inhibited by long-chain acyl-CoAs in a non-competitive manner, similar as previously shown for hormone-sensitive lipase (HSL), the rate-limiting enzyme for diglyceride breakdown in adipose tissue. ATGL activity is only marginally inhibited by medium-chain acyl-CoAs, diglycerides, monoglycerides, and free fatty acids. Immunoprecipitation assays revealed that acyl-CoAs do not disrupt the protein–protein interaction of ATGL and its co-activator CGI-58. Furthermore, inhibition of ATGL is independent of the presence of CGI-58 and occurs directly at the N-terminal patatin-like phospholipase domain of the enzyme. In conclusion, our results suggest that inhibition of the major lipolytic enzymes ATGL and HSL by long-chain acyl-CoAs could represent an effective feedback mechanism controlling lipolysis and protecting cells from lipotoxic concentrations of fatty acids and fatty acid-derived lipid metabolites.     

Antagonist minigenes identify genes regulated by parathyroid hormone through G protein-selective and G protein co-regulated mechanisms in osteoblastic cells

Parathyroid hormone (PTH) is the major hormone regulating bone remodeling. Binding of PTH to the PTH1 receptor (PTH1R), a heterotrimeric G protein coupled receptor (GPCR), can potentially trigger multiple signal transduction pathways mediated through several different G proteins. In this study, we employed G protein antagonist minigenes inhibiting Gα_s, Gα_q or Gα₁₂ to selectively dissect out which of these G proteins were responsible for effects of PTH(1-34) in targeted signaling and osteogenesis arrays consisting of 159 genes. Among the 32 genes significantly regulated by 24 h PTH treatment in UMR-106 osteoblastic cells, 9 genes were exclusively regulated through G_s, 6 genes were solely mediated through G_q, and 3 genes were only controlled through G₁₂. Such findings support the concept that there is some absolute specificity in downstream responses initiated at the G protein level following binding of PTH to the PTH1R. On the other hand, 6 PTH-regulated genes were regulated by both G_s and G_q, 3 genes were regulated by both G_s and G₁₂, and 3 genes were controlled by G_s, G_q and G₁₂. These findings indicate potential overlapping or sequential interactions among different G protein-mediated pathways. In addition, two PTH-regulated genes were not regulated through any of the G proteins examined, suggesting that additional signaling mechanisms may be involved. Selectivity was largely maintained over a 2-48-hour time period. The minigene effects were mimicked by downstream inhibitors. The dissection of the differential effects of multiple G protein pathways on gene regulation provides a more complete understanding of PTH signaling in osteoblastic cells.