2002 Global Researcher Conference Proceeding

April 26 - 28, 2002

Conference: 2002 Global Researcher Conference
Title: Quality control of nephrogenic diabetes insipidus-causing vasopressin V2 receptor mutants in the ER and in the ER/Golgi intermediate compartment
Authors: Hermosilla, Ricardo; Rosenthal, Walter; Schulein, Ralf
Institutions: Forschungsinstitut fur Molekulare Pharmakologie (FMP), Charite - Universitatsmedizin Berlin

The vasopressin V2 receptor (V2 receptor) is transported from the endoplasmic reticulum (ER) via the ER/Golgi intermediate compartment (ERGIC) and the Golgi apparatus to the plasma membrane. Nephrogenic diabetes insipidus (NDI)-causing mutations frequently lead to misfolded V2 receptors which are retained intracellularly by a quality control system which was thought to be located exclusively in the ER. Here we have examined whether quality control of the mutant receptors is indeed restricted to the ER or whether receptors may escape to the ERGIC and then be retrieved by the retrograde transport system of this compartment. To this end, the transport deficient NDI-causing V2 receptor mutants Y205C and the L62P were transiently expressed in HEK 293 cells. The retrograde transport from the ERGIC to the ER was then blocked with bafilomycin A1. At steady state conditions without bafilomycin treatment, both GFP-tagged mutants were detectable in the ER. Addition of bafilomycin A1 did not alter the localization of the L62P mutant indicating that this mutant never leaves the ER. The Y205C mutant, however, accumulated in the ERGIC upon bafilomycin A1 treatment as demonstrated by its colocalization with the ERGIC marker protein ERGIC53. The Y205C mutant thus normally reaches the ERGIC and then cycles back to the ER. Our results also demonstrate that different mutants of a membrane protein may be retained intracellularly by different quality control mechanisms.

The current model for the quality control of misfolded NDI-causing V2 receptors indicates that at least three components are involved: i) the calnexin/calreticulin system of the ER which recognizes the folding state of the receptors (Morello et al., 2001, Biochemistry 40, 6766-75), ii) an unknown system which seems to recognize exposed ER retention signals (RXR type) of misfolded forms (Hermosilla and Schülein, 2001, Mol Pharmacol. 60, 1031-39), and iii) the ERGIC which recognizes misfolded receptors which have escaped from the ER.

The vasopressin-2 receptor (V2R) protein is synthesized in the cell interior and must travel to the cell membrane to perform its function. It travels to the membrane by moving through cellular structures called the endoplasmic reticulum (ER), the ER/Golgi intermediate compartment (ERGIC) and the Golgi apparatus. Research indicated that V2Rs that, due to mutations, cannot achieve their proper shape, are retained in the ER. The ER exerts a quality control function, retaining those proteins improperly shaped (i.e. misfolded).

Schulein, et al., tested to see if the ER was the sole cellular structure involved in quality control of mutant receptor proteins. They did experiments with two V2R mutants. One mutant was indeed found only in the ER. The other mutant protein, however, could escape the ER only to be sent back to the ER by the ERGIC. Thus, different receptor protein mutants may be prevented from reaching the cell membrane by different quality control locations within the cell.