Abstract 499 Poster Session I, Saturday 5/1 (poster 307)

Naturally occurring mutants of the pituitary transcription factor Pit-1 cause combined pituitary hormone deficiency (CPHD) of growth hormone (GH), prolactin, and thyrotropin. In most autosomal recessive forms of CPHD, a Pit-1 mutation present on both alleles interferes with DNA binding resulting in disruption of activation and regulation of pituitary target genes. In sporadic and autosomal dominant forms of this disorder, however, Pit-1 mutations found on only one allele cause CPHD through dominant negative inhibition of wild type Pit-1 function. We have previously described a novel Pit-1 mutation, K216E, in a patient with hypopituitarism. Interestingly, however, this sporadic mutation does not decrease basal activation of human (h) GH gene expression.

The hGH promoter is regulated by Pit-1 and the protein kinase A (PKA) signaling pathway. Our data indicate that binding of Pit-1 to both Pit-1 binding sites in the hGH promoter is required for cyclic 3',5' monophosphate (cAMP) responsiveness. Pit-1 also interacts with two specific regions of the cAMP response element binding protein, CBP. In transient transfection studies, CBP acts as a transcriptional cofactor for wild type Pit-1 to permit synergistic activation of the hGH promoter by the PKA signaling pathway. However, the K216E mutant Pit-1 has decreased synergism with CBP of PKA-dependent activation of the hGH promoter. On the other hand, a mutant of Pit-1 lacking all its phosphorylation sites (S115A/T119A/T220A) is completely sufficient in activation of the hGH promoter, concluding that Pit-1 is not the target of phosphorylation by the PKA signaling pathway. Binding studies utilizing glutathione-S-transferase recombinant proteins reveal defective interaction between K216E mutant Pit-1 and CBP. Thus, our data indicate that a mechanism of hypopituitarism may be through impaired interaction between mutant Pit-1 and CBP.