Structural Analysis of GPS autoproteolysis of LNB-TM7 receptors – Characterization of the interaction between the extracellular α-subunit and the 7TM β-subunit

• Main Authors: Yi-Shu Huang, 黃益書
• Other Authors: Hsi-Hsien Lin
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/23985514582628849440

碩士 === 長庚大學 === 基礎醫學研究所 === 95 === The classical G protein-coupled receptors (GPCRs) control many physiological responses through one polypeptide chain, which normally consists of a 7TM domain and a short extracellular domain. In contrast, the novel class B2 GPCRs (LNB-TM7) have been found to undergo post-translational autoproteolysis at the G protein-coupled receptor proteolytic site (GPS) located within a conserved Cys-rich domain that is upstream of the first transmembrane segment. Therefore, the GPS autoproteolysis of LNB-TM7 receptors produces constitutively two subunits, a large extracellular domain (α-subunit) resembling cell- adhesion protein and a TM7 domain (β-subunit) that is a typical TM7 moiety of GPCR, from a single polypeptide chain in the endoplasmic reticulum soon after the receptor biosynthesis. The proteolytic mechanism is mediated by a self-catalyzed reaction commonly employed by the N-terminal nucleophile hydrolase style autoproteolysis. Intriguingly, the cleaved α-subunit was shown to remain firmly on the cell membrane. It has long been believed that the tight plasma membrane association is due to the non-covalent interaction with β-subunit. Hence, the LNB-TM7 heterdimer receptors are thought to function through ligand binding or protein-protein interaction via the α-subunit, followed by signal transduction via the β-subunit. In a recent study, Volynski et al. demonstrated that the α-subunit and β-subunit of latrophilin, a LNB-TM7 receptor for -latrotoxin, do not normally interact on the cell membrane, but behave rather as separate entities responsible for ligand binding and cellular signaling, respectively. However, the functional roles of GPS autoproteolysis and the fate of the α-subunit and β-subunit on the cell membrane remain to be identified. Using EMR2, a well characterized membrane of the EGF-TM7 family as a model molecule, we investigated the fates of the cleaved α-subunit and β-subunit. We now demonstrate that the α-subunit is likely anchored on the membrane directly by itself and the β-subunit is partially localized in the lipid raft. Nevertheless, separated α-subunit and β-subunit can re-associate in soluble form. Hence, we propose a model where the agonist binding to the α-subunit induces re-association to the β-subunit in the lipid raft and promotes signal transduction via the β-subunit.