CHARACTERIZATION OF HUMAN RETINOIC ACID RECEPTOR ALPHA-1 EXPRESSED INRECOMBINANT BACULOVIRUS-INFECTED SF9 INSECT CELLS

Full-length human retinoic acid receptor alpha 1 (hRAR alpha 1) was ex pressed in Sf9 insect cells using the baculovirus expression vector sy stem (BEVS). Western blot analysis using a specific anti-RAR peptide a ntiserum detected two major protein bands with apparent mol wts of sim ilar to 54 and similar to 51 kDa in extracts from insect cells infecte d with recombinant hRAR alpha 1 Autographica californica (AcNPV) bacul ovirus. Analysis of recombinant extracts from Sf9 cells labeled in viv o with [P-32]orthophosphate suggested that the recombinant protein was phosphorylated. A component in the recombinant nuclear extracts speci fically bound [H-3] all-trans-retinoic acid (RA) and sedimented in suc rose density gradient centrifugation as a single, symmetric peak with a sedimentation coefficient of similar to 3.6S, corresponding to a pro tein of approx 50 kDa. Scatchard analyses determined that [3H]RA was b ound in recombinant extracts by a single class of binding sites with a n apparent dissociation constant of similar to 0.3 nM and nuclear and cytoplasmic extracts contained similar to 1200 and similar to 200 pmol es, respectively, of unoccupied receptor per mg protein. In competitiv e ligand binding assays, relative binding affinities of 9-cis- and 13- cis-RA for hRAR alpha 1 in nuclear extracts were about threefold and s ixfold lower than all-trans-RA, whereas all-trans-retinol, -retinaldeh yde, and -retinyl acetate demonstrated relatively weak binding. In gel mobility shift assays, the electrophoretic migration of a [P-32]-labe led oligonucleotide containing the retinoic acid response element of t he RAR beta gene was retarded in the presence of recombinant nuclear a nd cytoplasmic extracts. The apparent complex formation between recomb inant hRAR alpha 1 and beta RARE was greatly enhanced by the addition of nuclear extract from wild-type AcNPV-infected Sf9 cells, possibly b ecause of heterodimer formation between recombinant hRAR alpha 1 and a metazoan RXR homolog. Thus, recombinant hRAR alpha 1 expressed at hig h levels in Sf9 insect cells exhibited biochemical properties of the n ative protein, including nuclear translocation, specific high affinity ligand and RARE binding, and possible heterodimer formation.