BADERC-Dennis Brown

Dennis Brown, Ph. D.

Regulation of protein/vesicle trafficking and polarity in epithelial cells

Dennis Brown is the Director of the MGH Program in Membrane Biology (PMB) which brings together investigators from several areas of research related to protein trafficking and cell signaling. The theme of the PMB is to understand how membrane transport vesicles interact with accessory proteins (ARFs, GTPases, SNAREs) and with the cytoskeleton (microtubules, actin, and PDZ proteins) in physiological and pathophysiological conditions. Experimental models used range from in vitro systems using purified proteins and membrane vesicles (endosomes, Golgi, plasma membrane), to transfected cell cultures, to whole animal models including transgenic mice. All projects are interactive and involve cell biological procedures such as confocal and EM-gold immunocytochemistry, organelle and membrane fractionation, immunoprecipitation, biotinylation, phosphorylation, pull-down assays, mass spectrometry and fluorescent tracer assays. Molecular procedures include preparation of adenovirus constructs, GST-fusion protein preparation, yeast two-hybrid screening, and real-time PCR. The aim of the Program is to understand how physiologically-relevant processes of fluid and electrolyte transport across epithelia are regulated at the cell and molecular levels. Representative projects include; 1) dissecting the mechanism by which water channel (aquaporin 2) trafficking occurs in renal epithelial cells in response to vasopressin. Kidney collecting duct principal cells respond to vasopressin by increasing the water permeability of their apical plasma membranes by exocytotic insertion of AQP2. We are examining the role of protein phosphorylation in this trafficking process using in vitro assays, transfected cell cultures, real-time microscopy, and in vivo animal studies; 2) understanding the pathways and proteins involved in the recycling of a vacuolar H⁺ATPase in renal epithelial cells in response to systemic acid/base changes. Specialized “coated” vesicles are involved in this process, but they contain no previously-identified coat proteins such as clathrin and caveolin. We are isolating and characterizing these unique transport vesicles to identify novel accessory proteins that could be involved in their trafficking; 3) identifying the mechanism by which Arf GTPases, exchange factors and other “coat”components are recruited to endosomes by intravesicular acidification. We are carrying out a proteomic screening of isolated vesicles to identify a putative “pH-sensing protein” on endosomal membranes that might be involved in coat recruitment after a luminal-pH-induced conformation change in its cytoplasmic domain.

These studies will help elucidate protein trafficking mechanisms in general, including those involved in glucose transporter trafficking.

References:

1. Breton, S., Wiederhold, T., Marshansky, V., Nsumu, N. N., Ramesh, V., and Brown, D. 2000. The B1 subunit of the H⁺ATPase is a PDZ-domain binding protein: colocalization with NHE-RF in renal B-intercalated cells. J. Biol. Chem., 275, 18219-18224.

2. Sullivan, B. M., Harrison-Lavoie, K. J., Marshansky, V., Lin, H. Y., Kehrl, J. H., Ausiello, D. A., Brown, D., and Druey, K. M. 2000. RGS4 binds coatomer and inhibits COPI association with Golgi membranes and intracellular transport. Mol. Biol. Cell.,11:3155-3168.

3. Maranda, B., Brown, D., Bourgoin, S., Casanova, J. E., Vinay, P., Ausiello. D. A. and Marshansky, V. 2001. Intraendosomal pH-sensitive recruitment of endogenous Arf6 and ARNO, but not Arf1, from cytoplasm to the endosomal membrane. J. Biol. Chem., 276: 18540-18550.

4. Sun, T-X., Van Hoek, A. N., Huang, Y., McLaughlin, M., and Brown, D. 2002. Aquaporin 2 localization in clathrin coated pits: inhibition of endocytosis by dominant negative dynamin. Am. J. Physiol. Renal Physiol., 282: F998-F1011.

5. Brown, D. The Ins and outs of aquaporin-2 trafficking. Am. J. Physiol. Renal Physiol. F893-F901, 2003.

6. Paunescu TG, Silva ND, Marshansky V, McKee M, Breton S, Brown D. Expression of the 56-kDa B2 subunit isoform of the vacuolar H+-ATPase in proton-secreting cells of the kidney and epididymis. Am J Physiol Cell Physiol. 2004;287:C149-62.

7. El-Annan J, Brown D, Breton S, Bourgoin S, Ausiello DA, Marshansky V. Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ. Am J Physiol Cell Physiol. 2004;286:C768-78.






			Dennis Brown, Ph. D. Regulation of protein/vesicle trafficking and polarity in epithelial cells Dennis Brown is the Director of the MGH Program in Membrane Biology (PMB) which brings together investigators from several areas of research related to protein trafficking and cell signaling. The theme of the PMB is to understand how membrane transport vesicles interact with accessory proteins (ARFs, GTPases, SNAREs) and with the cytoskeleton (microtubules, actin, and PDZ proteins) in physiological and pathophysiological conditions. Experimental models used range from in vitro systems using purified proteins and membrane vesicles (endosomes, Golgi, plasma membrane), to transfected cell cultures, to whole animal models including transgenic mice. All projects are interactive and involve cell biological procedures such as confocal and EM-gold immunocytochemistry, organelle and membrane fractionation, immunoprecipitation, biotinylation, phosphorylation, pull-down assays, mass spectrometry and fluorescent tracer assays. Molecular procedures include preparation of adenovirus constructs, GST-fusion protein preparation, yeast two-hybrid screening, and real-time PCR. The aim of the Program is to understand how physiologically-relevant processes of fluid and electrolyte transport across epithelia are regulated at the cell and molecular levels. Representative projects include; 1) dissecting the mechanism by which water channel (aquaporin 2) trafficking occurs in renal epithelial cells in response to vasopressin. Kidney collecting duct principal cells respond to vasopressin by increasing the water permeability of their apical plasma membranes by exocytotic insertion of AQP2. We are examining the role of protein phosphorylation in this trafficking process using in vitro assays, transfected cell cultures, real-time microscopy, and in vivo animal studies; 2) understanding the pathways and proteins involved in the recycling of a vacuolar H⁺ATPase in renal epithelial cells in response to systemic acid/base changes. Specialized “coated” vesicles are involved in this process, but they contain no previously-identified coat proteins such as clathrin and caveolin. We are isolating and characterizing these unique transport vesicles to identify novel accessory proteins that could be involved in their trafficking; 3) identifying the mechanism by which Arf GTPases, exchange factors and other “coat”components are recruited to endosomes by intravesicular acidification. We are carrying out a proteomic screening of isolated vesicles to identify a putative “pH-sensing protein” on endosomal membranes that might be involved in coat recruitment after a luminal-pH-induced conformation change in its cytoplasmic domain. These studies will help elucidate protein trafficking mechanisms in general, including those involved in glucose transporter trafficking. References: 1. Breton, S., Wiederhold, T., Marshansky, V., Nsumu, N. N., Ramesh, V., and Brown, D. 2000. The B1 subunit of the H⁺ATPase is a PDZ-domain binding protein: colocalization with NHE-RF in renal B-intercalated cells. J. Biol. Chem., 275, 18219-18224. 2. Sullivan, B. M., Harrison-Lavoie, K. J., Marshansky, V., Lin, H. Y., Kehrl, J. H., Ausiello, D. A., Brown, D., and Druey, K. M. 2000. RGS4 binds coatomer and inhibits COPI association with Golgi membranes and intracellular transport. Mol. Biol. Cell.,11:3155-3168. 3. Maranda, B., Brown, D., Bourgoin, S., Casanova, J. E., Vinay, P., Ausiello. D. A. and Marshansky, V. 2001. Intraendosomal pH-sensitive recruitment of endogenous Arf6 and ARNO, but not Arf1, from cytoplasm to the endosomal membrane. J. Biol. Chem., 276: 18540-18550. 4. Sun, T-X., Van Hoek, A. N., Huang, Y., McLaughlin, M., and Brown, D. 2002. Aquaporin 2 localization in clathrin coated pits: inhibition of endocytosis by dominant negative dynamin. Am. J. Physiol. Renal Physiol., 282: F998-F1011. 5. Brown, D. The Ins and outs of aquaporin-2 trafficking. Am. J. Physiol. Renal Physiol. F893-F901, 2003. 6. Paunescu TG, Silva ND, Marshansky V, McKee M, Breton S, Brown D. Expression of the 56-kDa B2 subunit isoform of the vacuolar H+-ATPase in proton-secreting cells of the kidney and epididymis. Am J Physiol Cell Physiol. 2004;287:C149-62. 7. El-Annan J, Brown D, Breton S, Bourgoin S, Ausiello DA, Marshansky V. Differential expression and targeting of endogenous Arf1 and Arf6 small GTPases in kidney epithelial cells in situ. Am J Physiol Cell Physiol. 2004;286:C768-78.