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

FEBS Letters

Hermosilla, Ricardo¹  Oksche, Alexander¹  Schülein, Ralf¹  Ebert, Jutta²  Lorenz, Dorothea¹  Rosenthal, Walter¹  Wiesner, Burkhard¹ 

[1]Forschungsinstitut für Molekulare Pharmakologie (FMP), Alfred-Kowalke-Str. 4, D-10315 Berlin, Germany

[2]Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig-Universität Giessen, Frankfurter Str. 107, D-35392 Giessen, Germany

关键词: Vasopressin V2 receptor;    Madin-Darby canine kidney cell;    Polarized transport;    Green fluorescent protein fusion;    BSA;    bovine serum albumin;    CCKAR;    cholecystokinin receptor type A;    FSH;    follicle-stimulating hormone;    GFP;    green fluorescent protein;    GPCR;    G protein-coupled receptor;    LH;    luteinizing hormone;    LSM;    laser scanning microscopy;    MDCK;    Madin-Darby canine kidney;    NBT;    nitro-blue tetrazolium;    PBS;    phosphate-buffered saline;    PhoA;    E. coli alkaline phosphatase;    PMSF;    phenylmethylsulfonyl fluoride;    TMB;    3;    3′;    5;    5′-tetramethylbenzamidine;    TSH;    thyrotropin;    V2 receptor;    vasopressin V2 receptor;   

DOI  :  10.1016/S0014-5793(98)01519-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We have analyzed the polarized cell surface expression of the G protein-coupled vasopressin V2 receptor (V2 receptor) in Madin-Darby canine kidney (MDCK) epithelial cells by both conventional cell surface biotinylation assays and laser scanning microscopy of green fluorescent protein (GFP)-tagged receptors. Cell surface biotinylation assays with stably transfected filter-grown cells expressing alkaline phosphatase (PhoA)-tagged receptors demonstrated that the V2 receptor is located predominantly basolaterally at steady state, while minor amounts are expressed apically. Laser scanning microscopy of filter- and glass-grown MDCK cells stably transfected with a GFP-tagged V2 receptor confirmed that the receptor is expressed mainly basolaterally; within the basolateral compartment, however, the receptor was confined to the lateral subdomain. The results obtained with the GFP-tagged receptor are thus consistent with and refine those from the biotinylation assay, which does not discriminate lateral from basal membrane regions. Our data indicate that the GFP methodology may effectively supplement cell surface biotinylation assays in future studies of polarized receptor transport. We finally 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 stably transfected cell lines, the experiments were complete within hours. The microinjection technique thus constitutes a powerful single cell technique to study the intracellular transport of G protein-coupled receptors. The methodology may be applicable to any cell type, even to tissue-derived, primary cultured cells; coinjection of transport-regulating compounds should also be possible.

【 授权许可】

Unknown