CONSTITUTIVELY ACTIVE G(S) ALPHA-SUBUNITS STIMULATE PIT-1 PROMOTER ACTIVITY VIA A PROTEIN-KINASE A-MEDIATED PATHWAY ACTING THROUGH DEOXYRIBONUCLEIC-ACID BINDING-SITES BOTH FOR PIT-1 AND FOR ADENOSINE-3',5'-MONOPHOSPHATE RESPONSE ELEMENT-BINDING PROTEIN
C. Gaiddon et al., CONSTITUTIVELY ACTIVE G(S) ALPHA-SUBUNITS STIMULATE PIT-1 PROMOTER ACTIVITY VIA A PROTEIN-KINASE A-MEDIATED PATHWAY ACTING THROUGH DEOXYRIBONUCLEIC-ACID BINDING-SITES BOTH FOR PIT-1 AND FOR ADENOSINE-3',5'-MONOPHOSPHATE RESPONSE ELEMENT-BINDING PROTEIN, Endocrinology, 137(4), 1996, pp. 1286-1291
Constitutively active mutations of the G protein alpha(S) subunit are
detected at a high frequency in human pituitary adenomas that secrete
GH or PRL. It seems possible that over-expression of the pituitary cel
l-specific transcription factor Pit-1/GHF-1 (Pit-1) gene in response t
o active alpha(S) subunits contributes to the formation of these adeno
mas. We have examined whether expression in pituitary cells of one of
these constitutively active alpha(S) subunits, Q227L-alpha(S), stimula
tes expression directed by the Pit-1 promoter. Transient expression of
Q227L-alpha(S) yielded a strong stimulation of a target Pit-1 promote
r-chloramphenicol acetyl transferase (CAT) construct, (-200)Pit-1-CAT.
Expression of wild-type alpha(S) or an inactive alpha(S) mutant yield
ed, respectively, reduced or no stimulation of CAT activity. A dominan
t inhibitor of protein kinase A (PKA), R(AB), blocked almost completel
y either forskolin (FSK) or Q227L-alpha(S) stimulation of (-200)Pit-1-
CAT expression, implying that PKA is required for the action of Q227L-
alpha(S) on the Pit-1 promoter. The Pit-1 promoter contains a binding
site for Pit-1 and two CREB binding sites. Mutation of the Pit-1 bindi
ng site reduced but did not eliminate either FSK or Q227L-alpha(S) sti
mulation of Pit-1 promoter activity, implying a partial but incomplete
requirement for this element for a PKA-mediated response to Q227L-alp
ha(S). The CREB dominant inhibitor S133A-CREB yielded a partial reduct
ion in either FSK or Q227L-alpha(S) stimulation of (-200)Pit-1-CAT exp
ression, implying that one or both of the Pit-1 promoter adenosine 3'5
'-monophosphate response element binding protein (CREB) binding sites
is/are also required for a complete PKA-mediated response to Q227L-alp
ha(S). The observation that S13SA-CREB completely blocked the response
to FSK or Q227L-alpha(S) of a Pit-1 promoter containing a mutated sit
e PitB1 implies that the binding sites for Pit-1 and CREB account for
all of the response elements for FSK or alpha(S) in the Pit-1 promoter
.