Membrane Targeting and Determination of Transmembrane Topology of the Human Vasopressin V2 Receptor

Title: Membrane Targeting and Determination of Transmembrane Topology of the Human Vasopressin V2 Receptor
Authors: Schulein, Ralf; Rutz, Claudia; Rosenthal, Walter
Publisher: Journal of Biological Chemistry
Date Published: November 15, 1996
Reference Number: 431
The human vasopressin V2 receptor belongs to the large family of G-protein-coupled receptors, which possess seven transmembrane helices, an extracellular N terminus and an intracellular C terminus. We have determined the sequence requirements of the V2 receptor for membrane insertion and correct topology for the inner membrane of Escherichia coli with the PhoA/LacZ gene fusion system. In addition, we have studied the signals for its membrane insertion and correct topology for the membrane of the endoplasmic reticulum of the authentic eucaryotic transport system. To this end, we have extended the PhoA/LacZ gene fusion system for the first time to eucaryotic cells, i.e. transiently transfected COS.M6 cells. Truncated V2 receptor sequences were fused to PhoA and LacZ and expressed in both E. coli and COS.M6 cells. Cells were fractionated, and LacZ/PhoA activity assays and immunoblots were performed. We show here that a V2 receptor fragment consisting of the N terminus, the first transmembrane segment and the first cytoplasmic loop (71 amino acids) provided sufficient information for membrane insertion and correct orientation (extracellular N terminus) in both procaryotic and eucaryotic cells. Our data differ substantially from those obtained for the human beta2-adrenergic receptor expressed in E. coli (Lacatena, R. M., Cellini, A., Scavizzi, F., and Tocchini-Valentini, G. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10521-10525). To establish correct topology, the beta2-adrenergic receptor requires a larger receptor portion, including the three N-terminal transmembrane segments and/or parts of the second cytoplasmic loop. The present data show that the observations made for the beta2-adrenergic receptor cannot be applied to G-protein-coupled receptors generally.
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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 human vasopressin-2 receptor (V2R) is a protein that functions in the membranes of specific cells, notably the principal cells of the kidney collecting duct. The V2R acts as a receptor for the antidiuretic hormone, arginine vasopressin (AVP). That is, the V2R binds with AVP, thereby initiating a molecular sequence that transmits the hormonal message of AVP.

V2Rs wait within the cell in their holding site, the endoplasmic reticulum (ER). When signaled, they leave the ER and travel to the cell membrane, a thin strip of tissue that encircles the cell, separating it from the environment. Once in the cell membrane they can bind with AVP. After it binds with AVP and AVP's message is transmitted, the V2R is either washed out of the body in the urine, or shuttles back to the ER.

Researchers have wondered what part of the V2R is responsible and necessary for getting it from the ER to the cell membrane. And, once there, what part of the V2R is responsible for orienting it correctly in relation to the cell membrane and the approaching AVP.

The V2R is a protein consisting of a string of 371 amino acids. When it is properly oriented in the cell membrane, the V2R has the following geometric configuration: the bulk of the V2R forms seven folded clumps within the cell membrane called transmembrane helices 1 - 7. Part of the V2R snakes outside the membrane to form three curves called extracellular loops 1 - 3. Part of it snakes inside the cell to form intracellular loops 1 - 3. One end of the V2R, called the amino- or N-terminus, sits outside the cell with the extracellular loops. The other end, called the carboxy-terminus, sits inside the cell with the intracellular loops. (You can look at a diagram of V2R here.)

Schulein, et al., determined the part of the V2R that inserts and properly orients the V2R in the inner membrane of the bacteria, Escherichia coli (E. coli). They then determined what part of the V2R was responsible for inserting and orienting the V2R in the membrane of the ER of the authentic eucaryotic transport system. (An eukaryotic cell is a cell that has a true nucleus. The authors created a specific eukaryotic cell culture within which they carried out their experiment.) The authors then compared the results of the two determinations.

The chose to work with E. coli because strategies have been developed to determine how membrane proteins insert and orient themselves in this bacteria. And also because G-protein coupled receptors (of which V2R is one) can be expressed in E. coli.

Schulein, et al., tracked the activity of the V2R in both the bacteria and eukaryotic cells by using the gene fusion system. Two enzymes, PhoA and LacZ were fused to truncated V2Rs, i.e. V2Rs that had portions of their receptors removed. PhoA is only active after it is transported across the inner membrane, whereas LacZ loses activity if the cell attempts to export it. By fusing these two enzymes onto truncated V2Rs and then analyzing the cell membranes for portions of PhoA and LacZ, the researchers were able to track and localize the V2R. In this way, they could find out which of the truncated receptors made its way into the membrane and properly oriented itself. (Proper orientation is crucial for proper binding with AVP.)

The authors determined the only part of the V2R that is needed to properly insert and orient itself in both the bacteria and the cell membrane is the first 71 amino acids. This includes the N-terminus, the first transmembrane helice, and the first intracellular loop.