Characterization of human retinoic acid receptor alpha 1 expressed in recombinant baculovirus-infected Sf9 insect cells.

Abstract

Full-length human retinoic acid receptor alpha 1 (hRAR alpha 1) was expressed in Sf9 insect cells using the baculovirus expression vector system (BEVS). Western blot analysis using a specific anti-RAR peptide antiserum detected two major protein bands with apparent mol wts of approximately 54 and approximately 51 kDa in extracts from insect cells infected with recombinant hRAR alpha 1 Autographica californica (AcNPV) baculovirus. Analysis of recombinant extracts from Sf9 cells labeled in vivo with [32P]orthophosphate suggested that the recombinant protein was phosphorylated. A component in the recombinant nuclear extracts specifically bound [3H]all-trans-retinoic acid (RA) and sedimented in sucrose density gradient centrifugation as a single, symmetric peak with a sedimentation coefficient of approximately 3.6S, corresponding to a protein of approx 50 kDa. Scatchard analyses determined that [3H]RA was bound in recombinant extracts by a single class of binding sites with an apparent dissociation constant of approximately 0.3 nM and nuclear and cytoplasmic extracts contained approximately 1200 and approximately 200 pmoles, respectively, of unoccupied receptor per mg protein. In competitive ligand binding assays, relative binding affinities of 9-cis- and 13-cis-RA for hRAR alpha 1 in nuclear extracts were about threefold and sixfold lower than all-trans-RA, whereas all-trans-retinol, -retinaldehyde, and -retinyl acetate demonstrated relatively weak binding. In gel mobility shift assays, the electrophoretic migration of a [32P]-labeled oligonucleotide containing the retinoic acid response element of the RAR beta gene was retarded in the presence of recombinant nuclear and cytoplasmic extracts. The apparent complex formation between recombinant hRAR alpha 1 and beta RARE was greatly enhanced by the addition of nuclear extract from wild-type AcNPV-infected Sf9 cells, possibly because of heterodimer formation between recombinant hRAR alpha 1 and a metazoan RXR homolog. Thus, recombinant hRAR alpha 1 expressed at high levels in Sf9 insect cells exhibited biochemical properties of the native protein, including nuclear translocation, specific high affinity ligand and RARE binding, and possible heterodimer formation.