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Variant Amino Acids in the Extracellular Loops of Murine and Human Vasopressin V2 Receptors Account for Differences in Cell Surface Expression and Ligand Affinity

Forschungsinstitut für Molekulare Pharmakologie (A.O., G.L., S.V., K.H., G.K., W.R.), D-13125 Berlin, Germany; Institut für Molekulare Biotechnologie Jena (M.P., S.G., A.R.), 07745 Jena, Germany; Fachhochschule Jena (S.G.), 07745 Jena, Germany; Friedrich-Schiller-Universität (A.R.), Biologisch-Pharmazeutische Fakultät, 07743 Jena, Germany; and Institut für Pharmakologie (W.R.), Freie Universität Berlin, 14195 Berlin, Germany

Address all correspondence and requests for reprints to: Alexander Oksche, Forschungsinstitut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany. E-mail: oksche{at}fmp-berlin.de.

Cloning and sequencing of the murine chromosomal region XB harboring the murine vasopressin V₂ receptor (mV₂R) gene and comparison with the orthologous human Xq28 region harboring the human vasopressin V₂ receptor (hV₂R) revealed conservation of the genomic organization and a high degree of sequence identity in the V₂R coding regions. Despite an identity of 87% of the amino acid sequences, both receptors show marked functional differences upon stable expression in Chinese hamster ovary cells: the mV₂R displayed a 5-fold higher affinity for [³H]AVP than the human ortholog; similar differences were found for the AVP-mediated activation of adenylyl cyclase. Saturation binding experiments with transiently transfected intact COS.M6 cells showed that the mV₂R was 3- to 5-fold less abundantly expressed at the cell surface than the hV₂R. Laser scanning microscopy of fusion proteins consisting of the V₂Rs and green fluorescent protein (GFP) (mV₂R/GFP, hV₂R/GFP) demonstrated that the hV₂R/GFP was efficiently transported to the plasma membrane, whereas the mV₂R/GFP was localized mainly within the endoplasmic reticulum. Chimeric hV₂Rs, in which the first and/or second extracellular loop(s) were replaced by the corresponding loop(s) of the mV₂R, revealed that the second extracellular loop accounts for the differences in ligand binding, but the first extracellular loop accounts for the reduced cell surface expression. The exchange of lysine 100 by aspartate in the first extracellular loop of hV₂R was sufficient to reduce cell surface expression, which was accompanied by intracellular retention as observed in laser scanning microscopy analysis. Conversely, the exchange of aspartate 100 by lysine in the mV₂R increased the cell surface expression and resulted in predominant plasma membrane localization. Thus, a single amino acid difference in the first extracellular loop between mV₂R and hV₂R determines the efficiency of cell surface expression.

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