Comparative analysis of the human angiotensin II type 1a receptor heterologously produced in insect cells and mammalian cells.
Angiotensin II type 1a receptor (AT1aR) is a member of GPCR superfamily and it plays crucial roles in the regulation of blood pressure, hormone secretion and renal functions. Here, we report functional overexpression and characterization of the human AT1aR in insect cells using the baculovirus system and in mammalian cells using the Semliki Forest virus system. The recombinant receptor was expressed at a level of 29-32 pmol/mg and it binds to angiontensin II with high affinity (Kd=0.98-1.1 nM). Angiotensin II stimulated accumulation of inositol phosphate and phosphorylation of MAP kinase was also observed, which indicated that the recombinant AT1aR could couple to endogenous Galphaq protein. Confocal laser scanning microscopy revealed that the recombinant receptor was predominantly localized in the plasma membrane and agonist induced internalization of the recombinant AT1aR was also observed. The recombinant AT1aR was expressed in glycosylated form and in vivo inhibition of glycosylation suppressed its surface expression.
Shukla
Arun Kumar
AK
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue Str. 3, 60438 Frankfurt am Main, Germany. Arun.Shukla@mpibp-frankfurt.mpg.de
Reinhart
Christoph
C
Michel
Hartmut
H
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D005821:Genetic Techniques; D006031:Glycosylation; D006801:Humans; D007313:Insecta; D018613:Microscopy, Confocal; D017434:Protein Structure, Tertiary; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D012672:Semliki forest virus / Q000235:genetics
D044140:Receptor, Angiotensin, Type 1; D011994:Recombinant Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11