Modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin.
Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing, inflammation, and host defense. PGRN also functions as a neurotrophic factor, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel, extracellular matrix protein, and growth factor. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor.
Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, USA.
Qing-Yun
QY
Tian
Yun-Peng
YP
Zhao
Chuan-ju
CJ
Liu
D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D036341:Intercellular Signaling Peptides and Proteins; D011498:Protein Precursors