Research

 

Our broad goal is to understand the human diseases caused by aberrant heterotrimeric G protein and GPCR signaling, so that clinical management of these diseases can be improved. Heterotrimeric G proteins mediate the actions of numerous hormones, neurotransmitters, and autocrine/paracrine factors. One of these proteins is the stimulatory G protein, which primarily acts by generating cyclic AMP (cAMP), a ubiquitous intracellular second messenger. The alpha-subunit of the stimulatory G protein (Gsα) has an intrinsic GTP hydrolase (GTPase) activity and is absolutely required during embryonic development. The loss of one copy of Gsα, resulting from genetic mutations, causes a variety of human disorders, such as Albright’s hereditary osteodystrophy, a disorder characterized by obesity, short-stature, brachydactyly, heterotopic ossification, and cognitive impairment.

Diminished Gsα levels/activity also leads to end-organ resistance to multiple hormones, such as parathyroid and thyroid stimulating hormone (pseudohypoparathyroidism). Enhanced activity of Gsα, which can also occur due to genetic mutations, is found in multiple benign and malignant tumors, such as pituitary adenomas and intraductal pancreatic mucinous neoplasms. Moreover, mutations that activate Gsα cause a complex endocrine disorder termed McCune-Albright Syndrome, characterized by fibrous dysplasia of bone, hyperpigmented skin lesions, and hyperactive endocrine organs.

The gene encoding Gsα is encoded by GNAS, a complex gene located on chromosome 20q13.3. The GNAS complex gene produces additional transcripts from both the sense and the antisense strand, and these transcripts are expressed either maternally or paternally due to the parent-of-origin specific imprinting of their promoters. Gsα itself is biallelically expressed in most tissues; however, the paternal Gsα allele is completely or partially silenced in a small set of tissues, such as renal proximal tubule, thyroid, pituitary, and certain parts of brain. Our current studies are focused on determining the roles of the different GNAS products, and the mutant forms thereof, in the pathophysiology of the diseases caused by inactivating or activating GNAS mutations.

- Cellular actions of the extra-large G alpha-protein (XLαs):

An important portion of our work entails the actions of the extra-large G protein, termed XLαs, which is identical to Gsα over the C-terminal portion comprising most of the functional elements necessary for G protein signaling. XLαs, however, has a unique N-terminal domain comprising multiple repeat motifs rich in prolines. We have shown that XLαs can mimic the actions of Gsα and is a more potent stimulator of cAMP generation at baseline than the latter. We showed that XLαs, unlike Gsα, escapes activation-induced subcellular redistribution. Our recent studies revealed that XLαs is able to activate signaling that is typically downstream of another G protein, i.e. Gq/11. In fact, rather than diminished Gs signaling, ablation of XLαs in mice is associated with diminished Gq/11 signaling, as determined by reduced intracellular production of inositol-1,4,5 trisphosphate (IP3) at baseline and in response to receptor stimulation. These studies identified XLαs as a unique G protein that can mimic both Gsα and Gq/11α. More recently, we performed a proteomic screen to identify novel interactors of XLαs, aiming to reveal its unique actions. This screen led to the discovery that XLαs forms complexes with sorting nexin-9 and dynamin, which are core components of clathrin-mediated endocytosis. Endocytosis is a fundamental cellular process that mediates the uptake of many nutrients and drugs and regulates cell signaling. After showing that XLαs is an inhibitor of endocytosis and iron uptake, we are now studying the molecular mechanisms underlying this action.

Our recent studies also aim at the role of XLαs in the regulation of mineral ion metabolism. Findings in some pediatric patients with GNAS mutations and mouse models indicate that XLαs actions are necessary for normal serum phosphate levels. Pursuing these findings through approaches that involve different mouse models, we discovered that XLαs mediates the renal actions of parathyroid hormone during early postnatal development. Furthermore, we found a role for XLαs in the production of the bone-derived phosphaturic hormone FGF23 during early postnatal period.  This role of XLas seems to be mediated, at least partly, by its action as a promoter of IP3/PKC signaling in a MAPK activation-dependent manner.

- Actions of the Gq/11 signaling pathway in FGF23 synthesis:

Our in vivo and in vitro studies have shown that the IP3/PKC signaling pathway is a novel mediator of FGF23 synthesis. We are currently investigating the downstream mechanisms and their interactions with the other known stimulators of FGF23 production, including phosphate and 1,25 dihydroxyvitamin D.  The study of G protein-coupled receptor(s) upstream of IP3/PKC signaling in this setting is also one of our important projects.  We are currently focuing on the receptors for lypophosphatidic acid, as a recent study by our collaborators in the Endocrine Unit has identified this agonist as a stimulator of FGF23 synthesis.

- Actions of the stimulatory G protein in kidney and bone and related genetic disorders:

The paternal Gsα allele is silenced in certain tissues, including the renal proximal tubule, and this imprinted expression profile determines the parental origin of the GNAS mutations leading to pseudohypoparathyroidism type-I. Mechanisms regulating the tissue-specific monoallelic expression of Gsα are poorly defined. Our investigations using laser-cut microdissection revealed that the monoallelic Gsα expression in the renal proximal tubule is temporally regulated during early postnatal development. This finding provided a plausible explanation for the latency of PTH resistance observed in patients with pseudohypoparathyroidism. Furthermore, we recently generated mice in which Gsα is ablated in the renal proximal tubule. Our results revealed the hormonal pathways contributing to the biochemical findings observed in patients with pseudohypoparathyroidism, implicating reduced 1,25 dihydroxyvitamin D levels in the pathogenesis.  We found that, rather than reduced synthesis, the loss of Gsα in the proximal tubule causes increased metabolism of this active form of vitamin D by leading to increased renal expression of Cyp24a1. 

- The role of activating GNAS mutations in skeletal development and disease:

We have also focused on the diseases caused by GNAS mutations that lead to constitutive activation of Gsα and XLαs. Our earlier studies using molecular methods and human samples from patients suggested that constitutive XLαs activity may contribute to the development of thyroid adenomas seen in patients with McCune-Albright Syndrome.  More recently, we generated a mouse model in which a Gsα mutant found in many patients with McCune-Albright Syndrome and Fibrous Dysplasia of Bone (Gsα-R201H) is expressed conditionally upon Cre recombinase. We have characterized the phenotype of these mice in which this mutant is expressed during limb development, revealing that constitutve Gsα signaling limits endochondral bone growth and results in short stature with an altered growth plate architecture.