William F. Schwindinger

Heterotrimeric G-protein βγ-dimers in growth and differentiation

Heterotrimeric G-proteins are components of the signal transduction pathways for the soluble and cell-contact signals that regulate normal growth and differentiation. There is now a greater appreciation of the role of the Gβγ-dimer in the regulation of a variety of intracellular effectors, including ion channels, adenylyl cyclase, and phospholipase Cβ. In many cases, Gβγ-dimers are required for the activation of mitogen activated protein kinase (MAPK) pathways that promote cellular proliferation, although the underlying mechanisms have yet to be fully elucidated. Activation of phosphotidylinositol-3-kinase (PI3K) is a critical step in the intracellular transduction of survival signals. Gβγ-dimers directly activate PI3Kγ as well as the more widely distributed PI3Kβ. The activation of PI3Kγ by Gβγ-dimers likely involves direct binding of specific Gβγ-dimers to both subunits of PI3Kγ. Thus, Gβγ-dimers transmit signals from numerous receptors to a variety of intracellular effectors in distinct cellular contexts. Five distinct Gβ-subunits and 12 distinct Gγ-subunits have been identified. New experimental approaches are needed to elucidate the specific roles of individual Gβγ-dimers in the pathways that transduce signals for proliferation and survival.

The cAMP–Protein Kinase A Signal Transduction Pathway Modulates Ethanol Consumption and Sedative Effects of Ethanol

Ethanol and other drugs of abuse modulate cAMP–PKA signaling within the mesolimbic reward pathway. To understand the role of the cAMP–PKA signal transduction in mediating the effects of ethanol, we have studied ethanol consumption and the sedative effects of ethanol in three lines of genetically modified mice. We report that mice with the targeted disruption of one Gsα allele as well as mice with reduced neuronal PKA activity have decreased alcohol consumption compared with their wild-type littermates. Genetic reduction of cAMP–PKA signaling also makes mice more sensitive to the sedative effects of ethanol, although plasma ethanol concentrations are unaffected. In contrast, mice with increased adenylyl cyclase activity resulting from the transgenic expression of a constitutively active form of Gsα in neurons within the forebrain are less sensitive to the sedative effects of ethanol. Thus, the cAMP–PKA signal transduction pathway is critical in modulating sensitivity to the sedative effects of ethanol as well as influencing alcohol consumption.

Clinical implications of genetic defects in G proteins. The molecular basis of McCune-Albright syndrome and Albright hereditary osteodystrophy.

Inactivating and activating mutations in the gene encoding G alpha s (GNAS1) are known to be the basis for 2 well-described contrasting clinical disorders, Albright hereditary osteodystrophy (AHO) and McCune-Albright syndrome (MAS). AHO is an autosomal dominant disorder due to germline mutations in GNAS1 that decrease expression or function of G alpha s protein. Loss of G alpha s function leads to tissue resistance to multiple hormones whose receptors couple to G alpha s. By contrast, MAS results from postzygotic somatic mutations in GNAS1 that lead to enhanced function of G alpha s protein. Acquisition of the activating mutation early in life leads to a more generalized distribution of the mosaicism and is associated with the classic clinical triad of polyostotic fibrous dysplasia, endocrine hyperfunction, and café au lait skin lesions described in MAS. Acquisition of a similar activating mutation in GNAS1 later in life presumably accounts for the restricted distribution of the gsp oncogene, and is associated with the development of isolated lesions (for example, fibrous dysplasia, pituitary or thyroid tumors) without other manifestations of MAS. Tissues that are affected by loss of G alpha s function in AHO are also affected by gain of G alpha s function in MAS, thus identifying specific tissues in which the second messenger cAMP plays a dominant role in cell growth, proliferation, or function. Further investigations of the functions of G alpha s and other members of the GTPase binding protein family will provide more insight into the pathogenesis and clinical manifestations of human disease.

Coupling of the PTH/PTHrP receptor to multiple G-proteins

Parathyroid hormone (PTH) elicits many of its physiological effects by activating distinct G-proteincoupled signaling cascades that lead to synthesis of cyclic AMP and hydrolysis of phosphatidylinositol 4,5-bisphosphate. Using the nonhydrolyzable photoreactive GTP analog [α-32P]GTP-γ-azidoanilide (GTP-AA) and peptide antisera raised against G-protein α-subunits, we studied coupling of the PTH receptor to G-proteins in rat osteoblast-like cells (ROS 17/2.8), and in human embryonal kidney cells expressing the cloned human PTH/parathyroid hormone-related peptide (PTHrP) receptor at 40,000 receptors/cell (C20) or 400,000 receptors/cell (C21). Incubation of C21 membranes (but not C20 membranes) with [Nle8,18, Tyr34]-bovine PTH(1-34) amide (bPTH[1-34]) led to concentration-dependent incorporation of GTP-AA into the two isoforms of Gαs, into Gαq/11, and to a much lesser extent into Gαi(1). In ROS 17/2.8 cells, bPTH(1-34) increased the incorporation of GTP-AA into Gαs, but not into Gαq/11 or Gαi. The ability of bPTH(1-34) to increase labeling of Gαs and Gαq/11 was correlated with the receptor-dependent sensitivity of the adenylyl cyclase and phospholipase C signaling pathways to the hormone.

Selective Resistance to Parathyroid Hormone Caused by a Novel Uncoupling Mutation in the Carboxyl Terminus of Gαs A CAUSE OF PSEUDOHYPOPARATHYROIDISM TYPE Ib

Gs is a heterotrimeric (α, β, and γ chains) G protein that couples heptahelical plasma membrane receptors to stimulation of adenylyl cyclase. Inactivation of one GNAS1 gene allele encoding the α chain of Gs (Gαs) causes pseudohypoparathyroidism type Ia. Affected subjects have resistance to parathyroid hormone (PTH) and other hormones that activate adenylyl cyclase plus somatic features termed Albright hereditary osteodystrophy. By contrast, subjects with pseudohypoparathyroidism type Ib have hormone resistance that is limited to PTH and lack Albright hereditary osteodystrophy. The molecular basis for pseudohypoparathyroidism type Ib is unknown. We analyzed theGNAS1 gene for mutations using polymerase chain reaction to amplify genomic DNA from three brothers with pseudohypoparathyroidism type Ib. We identified a novel heterozygous 3-base pair deletion causing loss of isoleucine 382 in the three affected boys and their clinically unaffected mother and maternal grandfather. This mutation was absent in other family members and 15 additional unrelated subjects with pseudohypoparathyroidism type Ib. To characterize the signaling properties of the mutant Gαs, we used site-directed mutagenesis to introduce the isoleucine 382 deletion into a wild type Gαs cDNA, transfected HEK293 cells with either wild type or mutant Gαs cDNA, plus cDNAs encoding heptahelical receptors for PTH, thyrotropic hormone, or luteinizing hormone, and we measured cAMP production in response to hormone stimulation. The mutant Gαs protein was unable to interact with the receptor for PTH but showed normal coupling to the other coexpressed heptahelical receptors. These results provide evidence of selective uncoupling of the mutant Gαs from PTH receptors and explain PTH-specific hormone resistance in these three brothers with pseudohypoparathyroidism type Ib. The absence of PTH resistance in the mother and maternal grandfather who carry the same mutation is consistent with current models of paternal imprinting of the GNAS1 gene.

Mice with Deficiency of G Protein γ3 Are Lean and Have Seizures

ABSTRACT Emerging evidence suggests that the γ subunit composition of an individual G protein contributes to the specificity of the hundreds of known receptor signaling pathways. Among the twelve γ subtypes, γ3 is abundantly and widely expressed in the brain. To identify specific functions and associations for γ3, a gene-targeting approach was used to produce mice lacking the Gng3 gene (Gng3−/−). Confirming the efficacy and specificity of gene targeting, Gng3 −/− mice show no detectable expression of the Gng3 gene, but expression of the divergently transcribed Bscl2 gene is not affected. Suggesting unique roles for γ3 in the brain, Gng3 −/− mice display increased susceptibility to seizures, reduced body weights, and decreased adiposity compared to their wild-type littermates. Predicting possible associations for γ3, these phenotypic changes are associated with significant reductions in β2 and αi3 subunit levels in certain regions of the brain. The finding that the Gng3 −/− mice and the previously reported Gng7 −/− mice display distinct phenotypes and different αβγ subunit associations supports the notion that even closely related γ subtypes, such as γ3 and γ7, perform unique functions in the context of the organism.

Adenosine A2a receptor signaling and Golf assembly show a specific requirement for the γ7 subtype in the striatum

The adenosine A2A receptor (A2AR) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein αolf subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ7 subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ7 protein controls the hierarchial assembly of a specific G-protein αolfβ2γ7 heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A2AR activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ7 subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A2AR G-protein αolfβ2γ7 interface as a possible therapeutic target for Parkinson disease.

Mice lacking the G protein γ3-subunit show resistance to opioids and diet induced obesity

Contributing to the obesity epidemic, there is increasing evidence that overconsumption of high-fat foods may be analogous to drug addiction in that the palatability of these foods is associated with activation of specific reward pathways in the brain. With this perspective, we report that mice lacking the G protein gamma(3)-subunit (Gng3(-/-) mice) show resistance to high-fat diet-induced weight gain over the course of a 12-wk study. Compared with Gng3(+/+) controls, female Gng3(-/-) mice exhibit a 40% reduction in weight gain and a 53% decrease in fat pad mass, whereas male Gng3(-/-) mice display an 18% reduction in weight gain and no significant decrease in fat pad mass. The basis for the lowered weight gain is related to reduced food consumption for female and male Gng3(-/-) mice of 13% and 14%, respectively. Female Gng3(-/-) mice also show a lesser preference for high-fat chow than their female Gng3(+/+) littermates, suggesting an attenuated effect on a reward pathway associated with overconsumption of fat. One possible candidate is the micro-opioid receptor (Oprm1) signaling cascade. Supporting a defect in this signaling pathway, Gng3(-/-) mice show marked reductions in both acute and chronic morphine responsiveness, as well as increases in endogenous opioid mRNA levels in reward-related regions of the brain. Taken together, these data suggest that the decreased weight gain of Gng3(-/-) mice may be related to a reduced rewarding effect of the high-fat diet resulting from a defect in Oprm1 signaling and loss of the G protein gamma(3)-subunit.

Heterotopic Ossifications in a Mouse Model of Albright Hereditary Osteodystrophy

Albright hereditary osteodystrophy (AHO) is characterized by short stature, brachydactyly, and often heterotopic ossifications that are typically subcutaneous. Subcutaneous ossifications (SCO) cause considerable morbidity in AHO with no effective treatment. AHO is caused by heterozygous inactivating mutations in those GNAS exons encoding the α-subunit of the stimulatory G protein (Gαs). When inherited maternally, these mutations are associated with obesity, cognitive impairment, and resistance to certain hormones that mediate their actions through G protein-coupled receptors, a condition termed pseudohypoparathyroidism type 1a (PHP1a). When inherited paternally, GNAS mutations cause only AHO but not hormonal resistance, termed pseudopseudohypoparathyroidism (PPHP). Mice with targeted disruption of exon 1 of Gnas (Gnas E1−/+) replicate human PHP1a or PPHP phenotypically and hormonally. However, SCO have not yet been reported in Gnas E1+/− mice, at least not those that had been analyzed by us up to 3 months of age. Here we now show that Gnas E1−/+ animals develop SCO over time. The ossified lesions increase in number and size and are uniformly detected in adult mice by one year of age. They are located in both the dermis, often in perifollicular areas, and the subcutis. These lesions are particularly prominent in skin prone to injury or pressure. The SCO comprise mature bone with evidence of mineral deposition and bone marrow elements. Superficial localization was confirmed by radiographic and computerized tomographic imaging. In situ hybridization of SCO lesions were positive for both osteonectin and osteopontin. Notably, the ossifications were much more extensive in males than females. Because Gnas E1−/+ mice develop SCO features that are similar to those observed in AHO patients, these animals provide a model system suitable for investigating pathogenic mechanisms involved in SCO formation and for developing novel therapeutics for heterotopic bone formation. Moreover, these mice provide a model with which to investigate the regulatory mechanisms of bone formation.

McCune-Albright syndrome

McCune-Albright syndrome (MAS) is characterized by the clinical triad of polyostotic fibrous dysplasia, cafe-au-lait pigmented skin lesions, and multiple endocrinopathies. The molecular basis of MAS is a mutation in G(s)alpha that results in constitutive activation of adenylyl cyclase in affected tissues. This mutation occurs during early embryogenesis, and therefore patients with MAS are mosaic. The identification of activating mutations of Gsa in liver, heart, and gastrointestinal tract of patients with MAS suggests a broader spectrum of clinical disease than previously appreciated.