SAT-546 Discovering the Function of IGSF1 and Its Role in the Hypothalamic-Pituitary-Thyroid Axis

Abstract

Loss of function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene cause congenital central hypothyroidism, hypoprolactinemia, macroorchidism, and the dysregulation of growth hormone and pubertal development. IGSF1 is a type 1 transmembrane protein of unknown function that is highly expressed in the pituitary gland, choroid plexus, and developing liver. The mature form of the protein contains seven extracellular Ig loops, a single transmembrane domain, and a short intracellular carboxyl tail rich in serine and threonine residues. Although the function of IGSF1 is presently unknown, the topology of the protein suggests that it plays a role in intercellular communication. Two Igsf1 knockout mouse models exhibit reduced pituitary TRH receptor (Trhr1) expression and associated impairments in TRH-stimulated TSH secretion. How the loss of IGSF1 leads to reductions in Trhr1 levels is unresolved. We hypothesize that IGSF1 acts in concert with intracellular signaling molecules to regulate Trhr1 expression. To date, IGSF1 has been shown to interact with only two proteins, itself and the thyroid hormone transporter MCT8. However, MCT8 has low expression in the pituitary and IGSF1 does not alter MCT8-dependent thyroid hormone transport. Therefore, we implemented a new proximity labelling method, BioID, to characterize the intracellular IGSF1 interactome and ultimately determine IGSF1’s role in Trhr1 regulation. We expressed an IGSF1-BirA* fusion protein in heterologous cells and confirmed that it properly traffics to the plasma membrane. Moreover, the protein auto-biotinylates and labels other, currently unidentified proteins. IGSF1-BirA* maintains its ability to interact with MCT8, but does not interact with an intracellular signaling protein recently implicated in central hypothyroidism, insulin receptor substrate 4 (IRS4). We are stably expressing IGSF1-BirA* in heterologous and homologous cell lines, HEK293 and HepG2 (human hepatocellular carcinoma), respectively, for the BioID screen. We will purify biotinylated proteins and identify them using mass spectrometry. In follow-up analyses, we will focus on proteins that link IGSF1 to known signaling pathways.