Polarized cell surface expression of the green fluorescent protein-tagged vasopressin V2 receptor in Madin Darby canine kidney cells

We have analyzed the polarized cell surface expression of the G protein-cou pled vasopressin V2 receptor (V2 receptor) in Madin-Darby canine kidney (MD CK) epithelial cells by both conventional cell surface biotinylation assays and laser scanning microscopy of green fluorescent protein (GFP)-tagged re ceptors, Cell surface biotinylation assays with stably transfected filter-g rown cells expressing alkaline phosphatase (PhoA)-tagged receptors demonstr ated that the V2 receptor is located predominantly basolaterally at steady state, while minor amounts are expressed apically. Laser scanning microscop y of filter- and glass-grown MDCK cells stably transfected with a GFP-tagge d V2 receptor confirmed that the receptor is expressed mainly basolaterally ; within the basolateral compartment, however, the receptor mas confined to the lateral subdomain, The results obtained with the GFP-tagged receptor a re thus consistent with and refine those from the biotinylation assay, whic h does not discriminate lateral from basal membrane regions. Our data indic ate that the GFP methodology may effectively supplement cell surface biotin ylation assays in future studies of polarized receptor transport. We finall y show that microinjection of a plasmid encoding the GFP-tagged V2 receptor into the nucleus of MDCK cells led to the same results as experiments with stably transfected cells. However, since there was no need for selecting s tably transfected cell lines, the experiments were complete within hours. T he microinjection technique thus constitutes a powerful single cell techniq ue to study the intracellular transport of G protein-coupled receptors, The methodology may be applicable to any cell type, even to tissue-derived, pr imary cultured cells; coinjection of transport-regulating compounds should also be possible, (C) 1998 Federation of European Biochemical Societies.