Prolatin-secreting lactotroph adenomas (prolactinomas), the most common human pituitary tumors, cause infertility, osteoporosis, loss of vision and loss of pituitary functions. Prolactin gene expression, tightly linked to lactotroph-specific function, is governed by Pit-1, a pituitary-specific POU- homeodomain transcription factor, that transduces cAMP/PKA and Ras signaling pathways. The Pit-1beta isoform contains the 26 amino-acid beta- domain, whose sequence specifies its role as a cell-type specific molecular switch. Pit-1beta blocks basal and Ras-stimulated prolactin promoter activity in GH4 pituitary cells, yet enhances basal and PKA-simulated prolactin promoter activity in HeLa non-pituitary cells. The goal of this proposal is to determine the precise molecular mechanism(s) by which the Pit-1 beta-domain dictates isoform-specific transcriptional responses. To this end, I plan to use molecular, biochemical and structural approaches to dissect the mechanistic structure-function relationships of the Pit-1 beta-domain, in the context of the distinct biological responses we have defined for Pit-1 versus Pit-1beta. I hypothesize that Pit-1 isoform-specific responses are dictated by specific amino acids brought to the Pit-1beta TAB by the beta domain insertion, and that the primary-structural changed created by the beta-domain induce three- dimensional structural changes in Pit-1/Pit-1beta, which may be responsible for the functional consequences of the beta-domain insertion. I came to UCHSC to join Dr. Gutierrez-Hartmann's laboratory in order to apply the molecular approaches to structure-function questions that I learned as a graduate student to the important field of molecular endocrinology. My work has lead to one publication, a review, a submitted manuscript and an internationally presented abstract on this exciting subject. This experience has helped to define an area of research of particular interest to me with potential for future independent development. Dr. Gutierrez-Hartmann's laboratory, where I will be carrying out the proposed work, is actively involved in molecular endocrinology research, and has an outstanding record of productivity. This award will allow me to advance my work on the Pit-1/Pit-1beta system so that I will be able to gain promotion to an independent tenure-tract position and to win the competitive funding necessary for me to successfully pursue that goal.
