MASSACHUSETTS GENERAL HOSPITAL, THE
Paradigm-shifting observations on aquaporin 2 (AQP2) and vasopressin receptor (V2R) trafficking, and vacuolar ATPase (V-ATPase) pH sensing and recycling have lead to new hypotheses to be addressed in this renewal. Project I will define VP-dependent and independent regulation of AQP2, and will identify proteins whose interaction with AQP2 is modified by phosphorylation to regulate trafficking. A novel role for AQP2 in regulating actin polymerization via interaction with RhoGAPS will be explored. Use of PDE5 inhibitors and statins to achieve VP-independent urine concentration will be tested as a potential strategy for future treatment of NDI. Project II will examine ligand induced conformational changes of the V2R at varying pH and tonicity using FRET techniques to dissect intra- and intermolecular protein interactions. Association of V2R with accessory proteins during internalization will be defined in cells expressing wild type and mutant V2R. Our novel observation that V2R interacts with the ESCRT protein Alix to accelerate V2R degradation will be pursued in these studies that address the regulation of body fluid homeostasis. Project III will pursue the breakthrough finding that the V-ATPase is an endosomal pH sensor by defining conformational changes in the V-ATPase tail that result in pH-dependent recruitment of small GTPases to membranes. It will identify pH sensitive residues on the luminal domains of the V-ATPase, and use albumin uptake to show relevance of the pH sensing mechanism to proximal tubule function. Project IV will elucidate downstream effectors (PKA, Epac) of soluble adenylate cyclase in modulating V-ATPase recycling and proton secretion in the epididymis, a "model" epithelium in which luminal acidic pH is critical for sperm maturation and storage. It will examine the role of cGMP-induced proton secretion in this tissue, and will address the exciting hypothesis that the V-ATPase is also an extracellular pH sensor that provides feedback control of luminal pH. These studies will allow a better understanding of male fertility, and uncover mechanisms that regulate V-ATPase in acidifying cells in general, including renal intercalated cells. The Microscopy Core B facility has been a major contributor to the success of this PPG. All projects gain considerable added value from extensive intellectual and technical collaborations that characterize our efforts to understand the relationship between cellular signaling, protein trafficking, and the responses of urogenital epithelial cells to their environment.
| AWARD OVERVIEW |
| Award Number |
3P01DK038452-23S1 |
Funding Agency |
Department of Health and Human Services |
| Total Award Amount |
$192,026 |
Project Location - City |
Boston |
| Award Date |
09/21/2009 |
Project Location - State |
MA |
| Project Status |
Completed |
Project Location - Zip |
02114-2696
|
| Jobs Reported |
0.00 |
Congressional District |
09 |
| Project Location - Country |
US |
|
|
Recipient Information
(Grants)
| Recipient Information (Grants) |
|
Recipient Name
|
MASSACHUSETTS GENERAL HOSPITAL, THE |
| Recipient DUNS Number |
073130411
|
| Recipient Address |
55 FRUIT ST |
| Recipient City |
BOSTON |
| Recipient State |
Massachusetts |
| Recipient Zip |
02114-2621 |
| Recipient Congressional District |
09 |
| Recipient Country |
USA |
Required to Report Top 5
Highly Compensated Officials |
No |
Projects and Jobs Information
| Projects and Jobs Information |
| Project Title |
Cellular Biology of Renal Function and Disease |
| Project Status |
Completed |
| Final Project Report Submitted |
Yes |
| Project Activities Description |
General Medical and Surgical Hospitals |
| Quarterly Activities/Project Description |
This will accelerate the pace of research in all four projects & increased the scientific impact of the projects by providing state-of-the-art technologies. We requested a Nikon Perfect Focus system that will allow greater & more effective utilization of our existing TIRF microscope for real time imaging by stabilizing the focus of the specimen over long time periods without human intervention, & a Leica FSP automated freeze-substitution & embedding system that will allow automated processing of tissue & cell samples for immuno-electron microscopy, which will greatly increase the throughput rate of EM samples provided to the Core for processing, as well as greatly enhancing our ability to detect antigenic sites by immunogold EM within these samples. Purchasing these will stimulate the economy by helping vendors & also because of follow-up spending on service, maintenance & disposables. The equipment will accelerate progress on our research by automating processes that are currently performed manually, often involving waiting times that are not productive for faculty & staff (manual focusing & reagent changing) The equipment will enhance the quality of scientific data by optimizing methods that are suboptimal (more precise image capture & analysis, better tissue & antigen preservation for EM gold labeling); Provide a safer working environment for PMB faculty & staff (reduced exposure to potentially harmful substances). It allowed us to retain an existing fully-trained technician, Zhenjie Jane Zhuang, who was supported by a 2 year local Pilot grant that expired. This experienced technician performed studies described in our application & has pushed the project forward to the extent that a new grant is now being submitted that will hopefully continue the employment of Zhuang, but will also allow us to hire 1 or 2 more personnel. The ARRA supplement achieved its goal of retention & allowed the work to move forward more quickly than would otherwise have been possible |
| Jobs Created |
0.00 |
| Description of Jobs Created |
This supplemental ARRA application was a) for the purchase of two major pieces of equipment for the Microscopy Core Facility of the PRogram in Membrane Biology and b) to maintain the employment of a trained technician, Zhenjie (Jane) Zhuang. a) Both of the requested pieces of equipment have been purchased and installed. The Leica FSP automated freeze substitution device was delivered in December and the purchase of this equipment obviously helps to sustain the activities of the vendor and all suppliers that provide materials and servicing of this equipment. The second piece of equipment, the Nikon Perfect Focus device, was delivered in late December and installed on January 4. Both pieces of equipment are in extensive daily use in the Microscopy Core Facility and are facilitating our work on this project as well as others. b) We are continuing our work on the function of a protein called a V-ATPase in endosomal/lysosomal protein degradative pathway, a fundamental pathway in all eukaryotic cells. Studies of its regulatory mechanisms are important for the design of novel approaches in order to control this pathway, as described in our proposal. In particular, we have synthesized various synthetic peptides derived from the so-called a2N subunit of the V-ATPase and have made siRNA and cDNA to regulate the function of an accessory protein called aldolase in cells. These reagents have been used in studies of trafficking and degradation of albumin within the endosomal/lysosomal protein degradative pathway in kidney cells, as well as on their action on the enzymatic activities of small GTPases, another type of interacting protein that regulates this pathway. As proposed, the funds have been used to retain our technician, Jane Zhenjie Zhuang who is working on this project. The results of these studies resulted in two articles which are currently under final stages of revision for their publication. She is the first author in author in one of these papers. |
Purchaser Information
(Grants)
| Purchaser Information |
| Contracting Office ID |
Not Reported |
| Contracting Office Name |
Not Available |
| Contracting Office Region |
Not Available |
| TAS Major Program |
75-0883 |
| Award Information |
| Award Date |
09/21/2009 |
| Award Number |
3P01DK038452-23S1 |
| Order Number |
|
| Award Type |
Grants |
| Funding Agency ID |
75 |
| Funding Agency Name |
Department of Health and Human Services |
| Funding Office Name |
Not Available |
| Awarding Agency ID |
75 |
| Awarding Agency Name |
Department of Health and Human Services |
| Amount of Award |
$192,026 |
| Funds Invoiced/Received |
$190,588 |
| Expenditure Amount |
$190,588 |
| Infrastructure Expenditure Amount |
$0 |
| Infrastructure Purpose and Rationale |
Not Reported |
| Infrastructure Point of Contact Name |
Not Reported |
| Infrastructure Point of Contact Email |
Not Reported |
| Infrastructure Point of Contact Phone |
Not Reported |
| Infrastructure Point of Contact Address |
Not Reported |
| Infrastructure Point of Contact City |
Not Reported |
| Infrastructure Point of Contact State |
Not Reported |
| Infrastructure Point of Contact Zip |
Not Reported |
Product or Service Information
(Grants)
| Product or Service Information |
| Primary Activity Code |
622110 |
| Activity Description |
General Medical and Surgical Hospitals |
| Sub-Awards Information |
| Sub-awards to Organizations |
0 |
| Sub-award Amounts to Organizations |
$0 |
| Sub-Awards to Individuals |
0 |
| Sub-Award Amounts to Individuals |
$0 |
| Number of Sub-awards less than $25,000/award |
0 |
| Amount of Sub-awards less than $25,000/award |
$0 |
| Number of payments to vendors greater than $25,000 |
0 |
| Total Amount of payments to vendors greater than $25,000/award |
$0 |
| Number of payments to vendors less than $25,000/award |
0 |
| Total Amount of payments to vendors less than $25,000/award |
$0 |
| Location Information |
| Latitude, Longitude |
42º 21' 44",
-71º 4' 11" |
| Congressional District |
09 |
| Address 1 |
55 Fruit Street |
| Address 2 |
|
| City |
Boston |
| County |
Suffolk |
| State |
MA |
| Zip |
02114-2696 |
|
 |