Overexpression and functional characterisation of the human melanocortin 4 receptor in Sf9 cells |
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Authors: | Dolby Viveka Collén Anna Lundqvist Andreas Cronet Philippe |
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Affiliation: | Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden. |
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Abstract: | 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. |
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Keywords: | Melanocortin receptor 4 GPCR Overproduction Insect cells Functional characterisation Membrane protein |
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