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Abstract

The ionophore monensin was found to markedly reduce the rate of return of vasopressin V₂-receptors to the membrane following down-regulation with [Arg⁸]vasopressin (AVP), as well as hormone dissociation (unloading) from cells following ligand binding and internalization in LLC-PK₁ renal epithelial cells. Monensin-resistant LLC-PK1 mutants were isolated and characterized for V₂-receptor recycling. Whilst the MN-41 mutant appeared to be impaired in [³H]AVP internalization, the MN-11 and MN-21 mutants exhibited parental V₂-receptor binding and internalization, but markedly impaired receptor recycling subsequent to ligand-dependent receptor down-regulation. Unloading subsequent to ligand binding and internalization at 37 degrees C was also much slower in the mutants either at 37 degrees C or 23 degrees C. In contrast, unloading subsequent to binding at 23 degrees C, or to binding at 37 degrees C in the presence of NH₄Cl, was comparable in LLC-PK1 and mutant cells implying the active nature of the recycling process impaired in the mutants. The mutations conferring resistance to monesin thus concomitantly impaired V₂-receptor recycling in the mutants. Results argue for a monensin-sensitive endosomal/lysosomal pathway for the renal V₂-receptor, representing the first such report for an adenylate cyclase stimulating receptor.