Ligand specificities of recombinant retinoic acid receptors RAR α and RAR β

Abstract

Binding of retinoic acid (RA) to specific RA receptors alpha and beta (RAR alpha and RAR beta) was studied. Receptors were obtained in two ways: (1) full-length receptors were produced by transient expression of the respective human cDNAs in COS 1 cells; and (2) the ligand-binding domains of RAR alpha and RAR beta were produced in Escherichia coli. RA binding to the wild-type and truncated forms of the receptor was identical for both RAR alpha and RAR beta, indicating that the ligand-binding domains have retained the binding characteristics of the intact receptors. Furthermore, RA bound with the same affinity to both RAR alpha and RAR beta. Only retinoid analogues with an acidic end-group were able to actively bind to both receptors. On measuring the binding of various retinoids, we have found that the properties of the ligand-binding sites of RAR alpha and RAR beta were rather similar. Two retinoid analogues were capable of binding preferentially to either RAR alpha or RAR beta, suggesting that it may be possible to synthesize specific ligands for RAR alpha and RAR beta.

In vivo biological activity of retinoids partially correlates to their affinity to recombinant retinoic‐acid receptor α and recombinant‐cellular retinoic‐acid‐binding protein I

“…Furthermore, retinoic acid is degraded and modified by specific and non-specific intracellular enzymic processes (Frolik, 1984;Gubler and Sherman, 1985;Williams and Napoli, 1985), which will possibly not accept the discussed synthetic retinoids. Recently, the affinities of Ro13-7410, TTAB, TTNN and Am580 to the recombinant ligandbinding domains of nuclear RAR were determined (Crettaz et al, 1990). Nevertheless, the relative binding affinities of these compounds, determined by Crettaz et al are , TTAB, TTNN, Am80 and Am580 in a chloramphenicol-acetyltransferase-receptor-activation assay (Lehmann et al, 1991 ;Graupner et al, 1991) are in agreement with those determined by the absorption assay in the present study.…”

Induction of the oxidative catabolism of retinoic acid in MCF-7 cells

“…In RbTE cells and HL-60 leukaemia cells, the biological activity of the synthetic retinoid CH55 was much higher (20-25 times) than that of all-trans-RA (Jetten et al, 1987). Overall, the RA catabolism-inducing capacity of the different retinoids correlates with the retinoic acid receptor binding affinities of the retinoids (Crettaz et al, 1990;Sani et al, 1990;Repa et al, 1993;Berggren Soderhund et al, 1995), suggesting that induction of RA catabolism is a receptor-mediated process. Two other lines of evidence substantiate this idea.…”

“…Induction of all-trans-RA catabolism was also obtained with other retinoids, CH55 >> 1 3-cis-RA = all-trans-RA > 9-cis-RA > 4-keto-all-trans-RA > 4-keto-1 3-cis-RA > retinol. The potency of the retinoids to induce all-trans-RA catabolism was correlated to their retinoic acid receptor affinity (Crettaz et al, 1990;Repa et al, 1990;Sani et al, 1990). Induction of all-trans-RA catabolism was inhibited by actinomycin D. Furthermore, all-trans-RA did not increase cytosolic retinoic acid-binding protein (CRABP) mRNA levels.…”

Retinoid Isomers Differ in the Ability to Induce Release of SMRT Corepressor from Retinoic Acid Receptor-α

“…Transient transfections were performed employing a liposome/ Lipofectin methodology (Life Technologies, Inc.), using 25 ng of pSG5 GAL4DBD vector, 100 ng of pSG5 GAL4AD vector, 100 ng of pGL2-GAL4 (17-mer) luciferase reporter plasmid, and 100 ng of a pCMVpromoter-lacZ reporter plasmid (used as an internal control) per well. The cells were subsequently incubated for 48 h in the presence or absence of all-trans-or 9-cis-retinoic acid and harvested, and the luciferase and ␤-galactosidase activities were determined as previously detailed (27,34,37,49).…”