Portal de Periódicos da CAPES

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

2002; Oxford University Press; Volume: 16; Issue: 4 Linguagem: Inglês

10.1210/mend.16.4.0796

1944-9917

A. Oksche, Gabriele Leder, Susanne Valet, Matthias Platzer, Kerstin Hasse, Soeren Geist, Gerd Krause, André Rosenthal, Walter Rosenthal,

Circadian rhythm and melatonin

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.