Recombinant Semliki Forest virus for over-expression and pharmacological characterisation of the histamine H(2) receptor in mammalian cells.
We describe the use of recombinant Semliki Forest virus (SFV) vectors for efficient expression of the rat histamine H(2) (rH(2)) receptor in COS-7 (African green monkey kidney cells) cells. Recombinant SFV-infected COS-7 cells express the histamine rH(2) receptor in a time-dependent fashion with a maximum expression level of 50 pmol mg(-1) after 40 h. SFV-mediated histamine rH(2) receptor expression shows similar pharmacological properties as the receptor expressed transiently or stably in mammalian cells. In addition, we demonstrate the pharmacological and functional characterisation of the D(115)N mutated histamine rH(2) receptor. It has been shown that the D(115)N mutation renders the receptor constitutively active and structurally unstable. The rapid onset of and high maximal expression levels obtained from SFV-infected COS-7 cells enabled us to characterise this mutant receptor. We prove that recombinant SFV vectors are powerful tools for heterologous expression of G-protein-coupled receptors and that one can achieve both the high-level gene expression described for baculovirus-infected insect cells and the use of mammalian cells as hosts.
Hoffmann
M
M
Leiden/Amsterdam Centre for Drug Research, Division of Medicinal Chemistry, Vrije Universiteit, FEW, De Boelelaan 1083, 1081 HV Amsterdam, Netherlands.
Verzijl
D
D
Lundstrom
K
K
Simmen
U
U
Alewijnse
A E
AE
Timmerman
H
H
Leurs
R
R
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D019556:COS Cells; D002460:Cell Line; D002927:Cimetidine / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D006146:Guanidines / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D007457:Iodine Radioisotopes; D009154:Mutation; D011869:Radioligand Assay; D011899:Ranitidine / Q000494:pharmacology; D051381:Rats; D011970:Receptors, Histamine H2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics*; D013997:Time Factors; D014162:Transfection
D006146:Guanidines; D007457:Iodine Radioisotopes; D011970:Receptors, Histamine H2; D011993:Recombinant Fusion Proteins; C063279:iodoaminopotentidine; D002927:Cimetidine; D006632:Histamine; D011899:Ranitidine; D000242:Cyclic AMP