Renal

Epithelial and Neuronal Cell Functions Mediated Through Modulation of Heterotrimeric G Proteins

Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce signals across cell membranes from receptors to a variety of intracellular effectors. Work from our laboratory has identified interactions of G protein a subunits with unique modulatory proteins expressed in neurons and epithelial cells. In addition to classical signaling in epithelial cells, we have identified important roles of G protein a subunits in regulating the epithelial cell junction. We have identified a direct interaction between Ga12 and ZO-1, a tight junction scaffolding protein, and showed that activated Ga12 disrupts junctional integrity through activation of Src tyrosine kinases and occludin may be a key Src target in these cells. Utilizing the yeast two hybrid interaction system and a human kidney library, we have identified an interaction between Ga12 and the A subunit of the serine-threonine phosphatase PP2A. We have now confirmed direct stimulation of PP2A activity by Ga12 (but not other Ga subunits) are defining the mechanism of activation and this pathway in regulating phosphorylation of target proteins such as the microtubule associated protein, tau in neurons and TJ proteins in MDCK cells.

The glomerular epithelial cell (podocyte) is a specialized polarized cell that forms multiple foot processes along the glomerular capillary basement membrane. The “junction” between foot processes (slit diaphragm) shares many features with traditional epithelial cell junctions and is essential for normal kidney function. Loss of podocyte slit diaphragms results in altered glomerular hemodynamics, proteinuria and progressive renal failure that is commonly seen in diabetic nephropathy. We have now established a functional assay for studying permeability in these cells and demonstrate regulated assembly of the barrier in a manner analogous to classical epithelial cell junctions. Furthermore, we are establishing establish transgenic mice with podocyte specific expression of activated Ga12 in addition to creating a Ga12 "knockin" mouse that will allow the role of these signaling pathways to be extended into disease models such as diabetic nephropathy.

Like epithelial cells, neurons are also highly polarized cells with restricted localization of G proteins into specialized membrane domains. We identified a unique interaction between pertussis toxin Ga subunits and a neuronal protein (Pcp2) exclusively expressed in cerebellar Purkinje cells. Pcp2 (Purkinje cell protein-2) regulates nucleotide exchange on Ga, but the functional implications for this interaction on Purkinje cell function have yet to be identified. Cell culture systems and biochemical approaches are being used to further define this interaction and the functional role in the cell. We find that Pcp2 stimulates neuronal differentiation and neurite growth through activation of Ras and p38 Map kinase pathways. Specialized cells such as neurons and epithelia utilize G proteins and novel interactions with regulatory proteins to provide essential cell-specific functions. Taken together, these studies will provide insights into neuronal and kidney cell functions that will lead to opportunities to modulate events in injured neurons and epithelia.

Recent Publications

1. Luo Y. and Denker, B.M. Interaction of Heterotrimeric G protein Gao with Purkinje Cell Protein-2: Evidence for a Novel Nucleotide Exchange Factor. 1999; J. Biol. Chem. 274:10685-10688.
   
   
2. Saha, C., Nigam, S.K. and Denker, B.M. Expanding Role of G Proteins in Tight Junction Regulation; Gas Stimulates TJ Assembly. 2001; Biochem. Biophys. Res. Comm. 285;250-256.
   
   
3. Meyer, T.N., Schwesinger, C., Denker, B.M. ZO-1 is a Scaffolding Protein for Signaling Molecules; Ga12 Directly Binds to the SH3 Domain and Regulates Paracellular Permeability in Epithelial Cells. 2002; J. Biol. Chem. 277:24855- 24858
   
   
4. Meyer, T.N., Schwesinger, C., Denker, B.M. G12 Regulates Paracellular Epithelial Cell Permeability Through Src Tyrosine Kinases. 2003; Am. J. Physiol. Cell Physiol 285;C1281-1293
   
   
5. Takeshi Yuasa, Ayumi Takakura, Bradley M. Denker, Bhuvarahamurthy Venugopal, and Jing Zhou. Polycystin-1L2 is a Novel G-protein Binding Protein. 2004; Genomics, 84;126-138.
   
   
6. Yao, J., Tu, C.L., Kos, C.H. Henderson, J.M., Allen, P.G., Denker, B.M., Pollak, M.R. a-actinin-4 mediated FSGS: an autosomal dominant kidney disease caused by a misfolded and rapidly degraded cytoskeletal protein. 2004; PLOS, 2;787-794.
   
   
7. Hunt, J., Pollak, M and Denker, BMCultured Podocytes Establish a Size Selective Barrier Regulated by Specific Signaling Pathways and Demonstrate Synchronized Barrier Assembly in a Calcium Switch Model of Junction Formation 2004, Submitted J.Am.Soc.Nephbr>
   
   
8. Zhu, D. Kosik, K.S., Yanamadala, V., and Denker, B.M.Ga12 Directly Interacts with PP2A; Evidence for Ga12-Stimulated PP2A Phosphatase Activity and Dephosphorylation of Microtubule Associated Protein, Tau. 2004, Submitted J. Biol. Chem
   
   
9. Guan, J., Luo??Y, and Denker, B.M. Purkinje Cell Protein-2 (Pcp2) Stimulates Neurite Differentiation and Growth in PC12 Cells Through Activation of P38 Map Kinase. 2004 Submitted to J Neurosci