Abstract
After agonist-induced internalization, the vasopressin V2 receptor (V2R) does not recycle to the plasma membrane. The ADP-ribosylation factor (ARF) proteins initiate vesicular intracellular traffic by promoting the recruitment of adaptor proteins; thus, we sought to determine whether ARF6 could promote V2R recycling. Neither the agonist-induced internalization nor the recycling of the V2R was regulated by ARF6, but a constitutively active mutant of ARF6 reduced cell-surface V2Rs 10-fold in the absence of agonist treatment. Visualization of the ARF6 mutant-expressing cells revealed a vacuolar-staining pattern of the V2R instead of the normal plasma membrane expression. Analysis of V2R maturation revealed that reduced cell-surface expression was due to the diminished ability of the newly synthesized receptor to migrate from the endoplasmic reticulum to the Golgi network. The same mechanism affected processing of the V1aR and acetylcholine M2 receptors. Therefore, ARF6 controls the exit of the V2 and other receptors from the endoplasmic reticulum in addition to its established role in the trafficking of plasma-membrane-derived vesicles.
Highlights
The vasopressin V2 receptor (V2R)2 is a member of the G-proteincoupled receptor (GPCR) superfamily of receptors that are characterized by seven membrane-spanning domains [1]
Active ARF6 Inhibits Cell-surface V2R Expression—We have previously shown that the mature wild-type V2R migrates as a diffuse band of 45–55 kDa on SDS-PAGE gels [21]
These pharmacological chaperones have been useful in attempting to delineate the mechanisms through which V2Rs are sorted to the plasma membrane after synthesis in the endoplasmic reticulum
Summary
The vasopressin V2 receptor (V2R) is a member of the G-proteincoupled receptor (GPCR) superfamily of receptors that are characterized by seven membrane-spanning domains [1]. Even though much of the T27N ARF6 protein aggregates intracellularly, a sufficient level of T27N ARF6 localizes to the plasma membrane where it acts as a dominant-negative inhibitor of ARF6 function by sequestering nucleotide exchange factors that activate ARF6 [17] These tools have been used to examine the ARF6 dependence of cell-surface protein trafficking. In the absence of agonist application, Q67L ARF6 reduced cell-surface V2R number by more than 10-fold, neither the agonist-induced internalization nor the recycling of the V2R was altered by the ARF6 constructs This is contrary to what happens with the TfnR, whereby TfnR levels at the plasma membrane were enhanced 2-fold [16], but similar to the effect of Q67L ARF6 on acetylcholine M2 receptor expression [18]. While attempting to delineate the route(s) of V2R trafficking affected by ARF6, we found a role for ARF6 in GPCR maturation
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