Pituitary Ets-1 and GABP bind to the growth factor regulatory sites of therat prolactin promoter

Ets factors play a critical role in oncogenic Ras- and growth factor-mediat ed regulation of the proximal rat prolactin (rPRL) promoter in pituitary ce lls. The rPRL promoter contains two key functional Ets binding sites (EBS): a composite EBS/Pit-1 element located at -212 and an EBS that co-localizes with the basal transcription element (BTE, or A-site) located at -96. Onco genic Ras exclusively signals to the -212 site, which we have named the Ras response element (RRE); whereas the response of multiple growth factors (F GFs, EGF, IGF, insulin and TRH) maps to both EBSs. Although Ets-1 and GA bi nding protein (GABP) have been implicated in the Ras and insulin responses, respectively, the precise identity of the pituitary Ets factors that speci fically bind to the RRE and BTE sites remains unknown. In order to identify the Ets factor(s) present in GH4 and GH3 nuclear extracts (GH4NE and GH3NE ) that bind to the EBSs contained in the RRE and BTE, we used EBS-RRE and B TE oligonucleotides in electrophoretic mobility shift assays (EMSAs), antib ody supershift assays, western blot analysis of partially purified fraction s and UV-crosslinking studies. EMSAs, using either the BTE or EBS-RRE probe s, identified a specific protein-DNA complex, designated complex A, which c ontains an Ets factor as determined by oligonucleotide competition studies. Using western blot analysis of GH3 nuclear proteins that bind to heparin-S epharose, we have shown that Ets-1 and GABP, which are MAP kinase substrate s, co-purify with complex A, and supershift analysis with specific antisera revealed that complex A contains Ets-l, GABP alpha and GABP beta1. In addi tion, we show that recombinant full-length Ets-1 binds equivalently to BTE and EBS-RRE probes, while recombinant GABP alpha/beta preferentially binds to the BTE probe. Furthermore, comparing the DNA binding of GH4NE containin g both Ets-1 and GABP and HeLa nuclear extracts devoid of Ets-1 but contain ing GABP, we were able to show that the EBS-RRE preferentially binds Ets-1, while the BTE binds both GABP and Ets-1. Finally, UV-crosslinking experime nts with radio-labeled EBS-RRE and BTE oligonucleotides showed that these p robes specifically bind to a protein of similar to 64 kDa, which is consist ent with binding to Ets-1 (54 kDa) and/or the DNA binding subunit of GABP, GABP alpha (57 kDa). These studies show that endogenous, pituitary-derived GABP and Ets-1 bind to the BTE, whereas Ets-1 preferentially binds to the E BS-RRE. Taken together, these data provide important insights into the mech anisms by which the combination of distinct Ets members and EBSs transduce differential growth factor responses.