Steroidogenic factor 1-DNA binding: a kinetic analysis using surface plasmon resonance

in Journal of Molecular Endocrinology
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Basal expression of the glycoprotein hormone alpha-subunit gene in pituitary gonadotrophs is partially dependent on a gonadotroph specific element (GSE) which binds the nuclear receptor, steroidogenic factor-1 (SF-1). We have used surface plasmon resonance (SPR) to determine the association (kappa ass), dissociation (kappa diss) and affinity (KA) constants of SF-1 binding to immobilized oligonucleotides containing either the GSE consensus motif or a GSE mutant with a 2 bp substitution in the GSE site (GSEMUT). In vitro translated SF-1 protein bound the consensus GSE with a threefold increase in affinity constant (P<0.01) compared with the GSEMUT. This was due primarily to a significant increase (P<0.05) in the kappa ass for SF-1 to the GSE and a slower kappa diss (P<0.05). The binding interaction was specific and could be significantly inhibited (P<0. 001) by either anti-SF-1 antibody or excess non-biotinylated GSE. The addition of 14 bp wild-type flanking sequences significantly reduced the affinity of SF-1 to both the GSE (P<0.05) and the GSEMUT (P<0.01). This was due to a significant (P<0.01) decrease in kappa ass for the wild-type and mutant long oligonucleotides compared with the short GSE. Nuclear extracts from alphaT3-1 gonadotroph cells also bound the GSE and GSEMUT, giving kappa diss values which were two- to threefold slower than those obtained with in vitro translated SF-1. Thus, SPR is a powerful technique for examining kinetic interaction between SF-1 and its binding site, and is able to demonstrate the effects of mutations and flanking sequences on that interaction.

 

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