Baculovirus-mediated expression of retinoic acid receptor type gamma in cultured insect cells reveals a difference in specific DNA-binding behavior with the 1,25-dihydroxyvitamin D3 receptor.
The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type gamma in Spodoptera frugiperda insect cells and Manduca sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of RAR gamma per mg of protein. The cytosolic preparation from infected insect cells exhibited an equilibrium dissociation constant of 2.1 nM as determined by a retinoic acid saturation analysis plotted by the method of Scatchard. A polyclonal antibody directed against RAR gamma recognized the recombinant receptor protein as a 54,000-Da species. Electrophoretic mobility shift analyses demonstrated that protein extracts from RAR gamma-producing insect cells or larvae are capable of retinoic acid responsive element binding. This contrasts with the specific DNA-binding behavior of the insect cell-produced vitamin D receptor, which requires the presence of a mammalian-derived nuclear accessory protein. This distinction between RAR gamma and the vitamin D receptor suggests a difference in the molecular requirements by these two receptors for specific binding of their respective DNA response elements.
Ross
T K
TK
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Prahl
J M
JM
Herzberg
I M
IM
DeLuca
H F
HF
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002117:Calcitriol / Q000378:metabolism; D002352:Carrier Proteins / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009036:Moths; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid / Q000378:metabolism*; D015183:Restriction Mapping; D014162:Transfection; D014212:Tretinoin / Q000378:metabolism
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid; D014212:Tretinoin; D002117:Calcitriol