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Aquaporin-2 localization in clathrin-coated pits: inhibition of endocytosis by dominant-negative dynamin.
Am J Physiol Renal Physiol. 2002 Jun; 282(6):F998-1011.AJ

Abstract

Before the identification of aquaporin (AQP) 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, by using a fracture-label technique applied to LLC-PK(1) 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 by using a GTPase-deficient dynamin mutation (K44A) to inhibit clathrin-mediated endocytosis. Immunofluorescence labeling and freeze-fracture electron microscopy 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 before internalization.

Authors+Show Affiliations

Program in Membrane Biology and Renal Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

11997316

Citation

Sun, Tian-Xiao, et al. "Aquaporin-2 Localization in Clathrin-coated Pits: Inhibition of Endocytosis By Dominant-negative Dynamin." American Journal of Physiology. Renal Physiology, vol. 282, no. 6, 2002, pp. F998-1011.
Sun TX, Van Hoek A, Huang Y, et al. Aquaporin-2 localization in clathrin-coated pits: inhibition of endocytosis by dominant-negative dynamin. Am J Physiol Renal Physiol. 2002;282(6):F998-1011.
Sun, T. X., Van Hoek, A., Huang, Y., Bouley, R., McLaughlin, M., & Brown, D. (2002). Aquaporin-2 localization in clathrin-coated pits: inhibition of endocytosis by dominant-negative dynamin. American Journal of Physiology. Renal Physiology, 282(6), F998-1011.
Sun TX, et al. Aquaporin-2 Localization in Clathrin-coated Pits: Inhibition of Endocytosis By Dominant-negative Dynamin. Am J Physiol Renal Physiol. 2002;282(6):F998-1011. PubMed PMID: 11997316.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Aquaporin-2 localization in clathrin-coated pits: inhibition of endocytosis by dominant-negative dynamin. AU - Sun,Tian-Xiao, AU - Van Hoek,Alfred, AU - Huang,Yan, AU - Bouley,Richard, AU - McLaughlin,Margaret, AU - Brown,Dennis, PY - 2002/5/9/pubmed PY - 2002/6/1/medline PY - 2002/5/9/entrez SP - F998 EP - 1011 JF - American journal of physiology. Renal physiology JO - Am. J. Physiol. Renal Physiol. VL - 282 IS - 6 N2 - Before the identification of aquaporin (AQP) 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, by using a fracture-label technique applied to LLC-PK(1) 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 by using a GTPase-deficient dynamin mutation (K44A) to inhibit clathrin-mediated endocytosis. Immunofluorescence labeling and freeze-fracture electron microscopy 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 before internalization. SN - 1931-857X UR - https://www.unboundmedicine.com/medline/citation/11997316/Aquaporin_2_localization_in_clathrin_coated_pits:_inhibition_of_endocytosis_by_dominant_negative_dynamin_ L2 - http://www.physiology.org/doi/full/10.1152/ajprenal.00257.2001?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -