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Articles in PresS, published online ahead of print December 11, 2001
Am J Physiol Renal Physiol, 10.1152/ajprenal.00257.2001
Submitted on August 15, 2001
Accepted on December 6, 2001
1 Program in Membrane Biology/Renal Unit, Massachusetts General Hospital, Boston, MA, USA
* To whom correspondence should be addressed. E-mail: ajpcell{at}rcn.com.
Prior to the identification of aquaporin proteins, vasopressin-regulated "water channels" were identified by freeze-fracture electron microscopy as aggregates or clusters of intramembraneous particles (IMPs) on hormonally-stimulated target cell membranes. In the kidney collecting duct, these IMP clusters were subsequently identified as possible sites of clathrin-coated pit formation on the plasma membrane, and a clathrin-mediated mechanism for internalization of vasopressin-sensitive water channels was suggested. Using an antibody raised against the extracellular C loop of AQP2, we now provide direct evidence that AQP2 is concentrated in clathrin coated pits on the apical surface of collecting duct principal cells. Furthermore, using a fracture-label technique applied to LLC-PK1 cells expressing an AQP2-c-myc construct, we show that AQP2 is located in IMP aggregates and is concentrated in shallow membrane invaginations on the surface of forskolin-stimulated cells. We also studied the functional role of clathrin-coated pits in AQP2 trafficking using a GTPase-deficient dynamin mutation (K44A), to inhibit clathrin-mediated endocytosis. Immunofluorescence labeling and freeze fracture EM showed that dominant-negative dynamin 1 and dynamin 2 mutants prevent the release of clathrin-coated pits from the plasma membrane, and induce an accumulation of AQP2 on the plasma membrane of AQP2-transfected cells. These data provide the first direct evidence that AQP2 is located in clathrin coated pits, and show that AQP2 recycles between the plasma membrane and intracellular vesicles via a dynamin-dependent endocytotic pathway. We propose that the IMP clusters previously-associated with vasopressin action represent sites of dynamin-dependent, clathrin-mediated endocytosis in which AQP2 is concentrated prior to internalization.
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