Overexpression and functional characterisation of the human melanocortin 4 receptor in Sf9 cells.
The human melanocortin 4 receptor (MC4r) was successfully expressed in Sf9 cells using the baculovirus infection system. N- and C-terminally His-tagged receptors generated B(max) values of 14 and 23 pmol receptor/mg membrane protein, respectively. The highest expression level obtained with the C-terminally His-tagged MC4r corresponded to 0.25mg active receptor/litre culture volume. Addition of a viral signal peptide at the N-terminus of the His-tagged MC4r did not improve the expression level. Confocal laser microscopy studies revealed that both the N- and C-terminally tagged MC4r did not accumulate intracellularly and were mainly located in the plasma membrane. The recombinant receptors showed similar affinity for the agonist NDP-MSH (Kd = 11 nM) as to MC4r expressed in mammalian cells. Functional coupling of the highest expressed C-terminal tagged receptor to endogenous Galpha protein was demonstrated through GTPgammaS binding upon agonist stimulation of the receptor. Ki values for the ligands MTII, HS014, alpha-, beta-, and gamma-MSH are comparable to the values obtained for MC4r expressed in mammalian cells.
Dolby
Viveka
V
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden.
Collén
Anna
A
Lundqvist
Andreas
A
Cronet
Philippe
P
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007313:Insecta; D007700:Kinetics; D007834:Lasers; D008024:Ligands; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044105:Receptor, Melanocortin, Type 4 / Q000096:biosynthesis* / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
D017931:DNA Primers; D008024:Ligands; D044105:Receptor, Melanocortin, Type 4; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate)