2000 Global Researcher Conference Proceeding
March 10 - 12, 2000
|Conference:||2000 Global Researcher Conference|
|Title:||V2 Vasopressin Receptors in NDI: Where did they go?|
|Authors:||Wildin, Robert; Filanoski, Brian J.; Green, Lindsay; Cogdell, David E.|
|Institutions:||(formerly) Oregon Health Sciences University, MP350, Oregon Health Sciences University|
Background: The definition of NDI is renal hypo- or un-responsiveness to vasopressin. (AVP). X-linked NDI is due to defects in the AVPR2 gene encoding the V2 vasopressin receptor (V2R). The molecular and cellular disposition of several mutant receptors has been examined, and a variety of molecular variations identified. We sought to expand that knowledge by studying additional mutants in detail.
Methods: AVPR2 genes from 16 patients with NDI were cloned and expressed in cultured cells. Residual signaling activity was assessed in JEG3 cells as previously reported. [3H]AVP binding to transiently transfected, intact COS-7 cells was determined using standard saturating binding techniques. Surface expression was assessed by immunofluorescence or cell-based ELISA in COS-7 cells using plasmids with a Hemagglutinin epitope tag engineered into the N-terminus of the receptor sequence (nHA). Subcellular distribution was determined using antiserum recognizing the center of the third cytoplasmic loop of the V2R. Co-immunostaining was performed for a number of cell components and organelles. To assess the proportion of synthesized protein that encounters quality control in the ER and is degraded, cells were treated with proteasome inhibitors.
Results: No NDI receptor signaled at AVP concentrations found in vivo. Six of 16 had partial or complete signaling capability with more AVP. Of those, 4 had detectable binding on the cell surface, though two had markedly reduced affinity. Eight of 16 mutants had faint to full surface expression, while the rest did not reach the surface. Four of the 5 with high surface expression had nearly normal intracellular distribution, but the rest showed clumps and/or ropes. In many an unusual arc-shaped juxtanuclear “rind” of V2R was observed. Further investigation revealed that it shares many characteristics with the aggresome, first described with CFTR: microtubule dependence, co-localization with microtubule organizing center, enhancement by proteasome inhibitors, and rearrangement of intermediate filaments. The exact localization of mutant V2R, however, is different from CFTR.
Conclusions: The majority of AVPR2 mutations causing NDI are deemed misfolded by the ER quality control system and redirected for degradation by the proteasome. Failure to be expressed on the cell surface is the proximate cause of AVP unresponsiveness and is epistatic to binding or signaling defects. Mutant V2R has the capacity to overwhelm the proteasome system in vitro and to induce spontaneous aggresomes. Loss of function mutations reaching the cell surface include non-conservative changes in TM7, as well as alterations in extracellular residues, while mutations from TMs 1-6 are generally surface incompetent.