Appropriate Polarization Following Pharmacological Rescue of V2 Vasopressin Receptors Encoded by X-Linked Nephrogenic Diabetes Insipidus Alleles Involves a Conformation of the Receptor That Also Attains Mature Glycosylation

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Title: Appropriate Polarization Following Pharmacological Rescue of V2 Vasopressin Receptors Encoded by X-Linked Nephrogenic Diabetes Insipidus Alleles Involves a Conformation of the Receptor That Also Attains Mature Glycosylation
Authors: Tan, C.M.; Nickols, Hilary Highfield; Limbird, Lee E.
Publisher: Journal of Biological Chemistry
Date Published: September 12, 2003
Reference Number: 615
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To understand the mechanisms of G protein-coupled receptor (GPCR) delivery and steady state localization, we examined the trafficking itineraries of wild type (WT) and mutant V2 vasopressin receptors (V2R) in polarized Madin-Darby Canine Kidney II (MDCK II) cells and in COS M6 cells; the mutant V2Rs selected represent alleles responsible for X-linked nephrogenic diabetes insipidus. The WT V2R is localized on the plasma membrane and mediates AVP-stimulated cAMP accumulation, whereas the clinically relevant V2R mutants, L292P V2R, deltaV278 V2R, and R337X V2R, are retained intracellularly, are insensitive to extracellularly added AVP, and are not processed beyond initial immature glycosylation, manifest by their endoglycosidase H sensitivity. Reduced temperature and pharmacological, but not chemical, strategies rescue mutant V2Rs to the cell surface of COS M6 cells; surface rescue of L292P V2R and R337X V2R, but not of deltaV278 V2R, parallels acquisition of AVP-stimulated adenylyl cyclase activity. Pharmacological rescue of the L292P or R337X V2R by incubation with the membrane-permeant V2R antagonist, SR121463B, leads to a mature glycosylated form of the receptor that achieves localization on the basolateral surface of polarized MDCK II cells indistinguishable from that of the WT V2R. Surprisingly, however, the immature form of the mutant L292P V2R escapes to the apical, but not basolateral, surface of polarized MDCK II cells, even in the absence of SR121463B. These findings are consistent with the interpretation that the receptor conformation that allows appropriate processing through the N-linked glycosylation pathway is also essential for targeting to the appropriate surface of polarized epithelial cells.

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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

The vasopressin 2 receptor (V2R) protein is synthesized within the principal cells of the kidney collecting duct. To perform its function of linking with the hormone, vasopressin (AVP), in order to initiate the molecular process that results in urine concentration, V2R must travel from the cell interior to a section of the cell membrane known as the basolateral membrane. The vast majority of cases of NDI are due to mutations of the V2R gene that result in mutated V2R proteins. Over 70% of the mutations synthesize misshapen V2Rs that are unable to travel from within the cell interior to the basolateral membrane. V2Rs unable to reach the membrane are unable to link with AVP, as AVP cannot travel to the cell interior.

Tan, et al., traced the movements of normal V2Rs and three NDI-causing V2R mutants (L292P, DV278, and R337X) in two different types of laboratory cell cultures to better understand how they were either able or unable to travel to the basolateral membrane. The researchers found that the three mutant V2Rs were clearly trapped within the cell interior. However, they found that if they incubated one of the cell cultures (COS M6) at 28 degrees C, a significant amount of the mutant V2Rs were able to reach the cell membrane. That means these mutant V2Rs are temperature sensitive. The researchers speculate that a reduction of the temperature at which the V2R are grown somehow promotes the proper V2R shape, thus allowing the V2Rs to pall the cell?s quality control system and travel to the cell surface.

Tan, et al., also found that they could rescue the mutant V2Rs, i.e., manipulate them so that a significant number can get to the cell surface, by adding the substance SR121463B to the cell culture. This substance is able to get through the cell membrane and into the cell interior where it binds with the V2R.

Once the researchers found that mutant V2Rs could be induced to the membrane pharmacologically as well as through temperature manipulation, they tested to see if the rescued mutants could function. That is, they tested as to whether the rescued mutant V2Rs could bind with AVP and initiate the molecular sequence leading to urine concentration. The mutant DV278 could not function once it arrived at the cell membrane. However, L292P and R337X could bind with AVP and initiate the molecular process, though not to the degree a normal V2R could.

Glycosylation is a chemical process where a molecular structure, such as a protein, forms a link with a glycosyl group. A V2R that achieves glycosylation is considered mature. The mutant V2Rs trapped within the cell were immature glycosylated forms of the protein, and therefore susceptible to dismantling by enzymes within the cell. When these mutants are rescued pharmacologically, they not only travel to the basolateral membrane, they achieve mature glycosylation. The finding is that appropriate delivery to the basolateral surface involves reshaping the mutant V22R so that it is capable of mature glycosylation.

The researchers conclude that for NDI patients with mutants L292P and R337X, SR121463B and other agents like it (i.e., agents that are able to penetrate the cell membrane and bind with V2R) may act as a pharmacological rescue, enabling the V2R to get to the basolateral membrane and, once there, bind with AVP.