Molecular cloning, stable expression and cellular localization of human alpha1-adrenergic receptor subtypes: effect of charcoal/dextran treated serum on expression and localization of alpha1D -adrenergic receptor.
The cDNAs encoding for three subtypes of adrenergic receptors, alpha1A-, alpha1B- and alpha1D-ARs, were cloned and expressed in HEK 293 cells. Expression of alpha1A- and alpha1B-AR subtypes in HEK 293 cells was stable even with increased passages but that of alpha1D-AR was not. Cellular localization studies using immunofluorescence and flow cytometry revealed that expression of alpha1A- and alpha1B-ARs was primarily localized on the cell membrane whereas expression of alpha1D-AR was predominantly intracellular. Our studies clearly demonstrated that the culturing of the recombinant cell lines expressing alpha1D-AR in charcoal/dextran treated fetal bovine serum (FBS) resulted in targeting of alpha1D-AR to the cell membrane and thus, significantly improving its stability and availability for ligand binding studies.
Khattar
Sunil K
SK
Department of Biotechnology, Ranbaxy Research Laboratories, Plot No. 20, Sector 18, Udyog Vihar Industrial Area, Gurgaon, 122001 Haryana, India. sunil.khattar@ranbaxy.com
Bora
Roop Singh
RS
Priyadarsiny
Priyanka
P
Gautam
Aarti
A
Gupta
Dikshi
D
Tiwari
Atul
A
Nanda
Kamna
K
Singh
Rahul
R
Chugh
Anita
A
Bansal
Vinay
V
Mookhtiar
Kasim
K
Saini
Kulvinder Singh
KS
D018929:Cell Culture Techniques; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002606:Charcoal / Q000494:pharmacology; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry* / Q000494:pharmacology*; D018076:DNA, Complementary / Q000302:isolation & purification; D003911:Dextrans / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology; D005312:Fetal Blood / Q000737:chemistry; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007668:Kidney / Q000166:cytology; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D044967:Serum / Q000737:chemistry
C547713:ADRA1A protein, human; C547716:ADRA1B protein, human; C547719:ADRA1D protein, human; D003470:Culture Media; D018076:DNA, Complementary; D003911:Dextrans; D018340:Receptors, Adrenergic, alpha-1; D011994:Recombinant Proteins; D002606:Charcoal