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

Abstract

Basal expression of the glycoprotein hormone α-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 (k(ass)), dissociation (k(diss)) and affinity (K(A)) 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 (GSE(MUT)). In vitro translated SF-1 protein bound the consensus GSE with a threefold increase in affinity constant (P < 0.01) compared with the GSE(MUT). This was due primarily to a significant increase (P < 0.05) in the k(ass) for SF-1 to the GSE and a slower k(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 GSE(MUT) (P < 0.01). This was due to a significant (P < 0.01) decrease in k(ass) for the wild-type and mutant long oligonucleotides compared with the short GSE. Nuclear extracts from αT3-1 gonadotroph cells also bound the GSE and GSE(MUT) giving k(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.