Splicing and Multiple Binding Proteins in the Corticotropin-Releasing Hormone Stress System.

Abstract

Corticotropin-releasing hormone (CRH) is an important mediator of the mammalian stress response. Functioning both as a neurotransmitter and endocrine hormone, it signals through two receptors, CRH-R1 and CRH-R2. CRH is also bound with high-affinity by CRH-Binding Protein (CRH-BP), a secreted glycoprotein. As soluble binding proteins can play an important role in modulating the availability and activity of ligands at the receptors, this thesis focuses on the characterization of multiple binding proteins in the CRH system, including both truncated splice forms of CRH-R2 and the classical CRH-BP. First, we identified splice variants of CRH-R2 that were predicted to serve as soluble-decoy receptors because they encode the extracellular, ligand-binding domain of CRH-R2 but terminate prior to the transmembrane domains. These splice variants, called soluble CRH-R2 (sCRH-R2) α and β, encode similar proteins but have unique N-termini. We demonstrated that the α isoform of sCRH-R2 was efficiently translated in vivo, despite being a predicted substrate for nonsense-mediated RNA decay; however, the resulting protein was not trafficked for secretion due to an ineffective signal peptide, and was consequently degraded by the proteasome. In contrast, the β isoform of sCRH-R2, with its unique signal peptide, was properly trafficked for secretion and escaped degradation. Therefore, unlike sCRH-R2α, sCRH-R2β is positioned to function as a soluble CRH-binding protein. For analysis of CRH-BP, current models suggest that CRH-BP and CRH-receptors compete for available ligand, yet the rate of ligand association and dissociation, which are paramount to this competition, were not established. We determined these kinetic parameters for CRH with CRH-BP and CRH-R2, showing that CRH binds faster and releases more slowly for CRH-BP, which suggests CRH-BP is an efficient ligand trap. Furthermore, we demonstrated that CRH-BP inhibits CRH-R2 activation in cell culture. Strikingly, the amplitude and duration of the inhibition was dependent on the time of ligand interaction with CRH-BP prior to encountering the receptors, highlighting the importance of kinetic and temporal considerations in defining the function of CRH-BP. Together, these studies further characterize the role of both the classic and alternative binding proteins in the CRH system and advance our understanding of their function in stress biology.