Biochem. J. (2008) 414
(399–406) (Printed in Great Britain)
Characterization of the topology and functional domains of RKTG
Xiaolin LUO*, Lin FENG*, Xiaomeng JIANG*, Fei XIAO*, Zhenzhen WANG*, Gen-Sheng FENG† and Yan CHEN*1
*Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China, and †Program in Signal Transduction, Burnham Institute for Medical Research, La Jolla, California 92037, U.S.A.
RKTG (Raf kinase trapping to Golgi) is exclusively localized at the Golgi apparatus and functions as a spatial regulator of Raf-1 kinase by sequestrating Raf-1 to the Golgi. Based on the structural similarity with adiponectin receptors, RKTG was predicted to be a seven-transmembrane protein with a cytosolic N-terminus, distinct from classical GPCRs (G-protein-coupled receptors). We analysed in detail the topology and functional domains of RKTG in this study. We determined that the N-terminus of RKTG is localized on the cytosolic side. Two short stretches of amino acid sequences at the membrane proximal to the N- and C-termini (amino acids 61–71 and 299–303 respectively) were indispensable for Golgi localization of RKTG, but were not required for the interaction with Raf-1. The three loops facing the cytosol between the transmembrane domains had different roles in Golgi localization and Raf-1 interaction. While the first cytosolic loop was only important for Golgi localization, the third cytosolic loop was necessary for both Golgi localization and Raf-1 sequestration. Taken together, these findings suggest that RKTG is a type III membrane protein with its N-terminus facing the cytosol and multiple sequences are responsible for its localization at the Golgi apparatus and Raf-1 interaction. As RKTG is the first discovered Golgi protein with seven transmembrane domains, the knowledge derived from this study would not only provide structural information about the protein, but also pave the way for future characterization of the unique functions of RKTG in the regulation of cell signalling.
Key words: Golgi apparatus, progesterone and adipoQ receptor (PAQR), Raf-1, RKTG.
Abbreviations used: aa, amino acid; EGFP, enhanced GFP (green fluorescent protein); ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; GPCR, G-protein-coupled receptor; HA, haemagglutinin; HEK-293T, HEK-293 cells (human embryonic kidney cells) expressing the large T-antigen of SV40 (simian virus 40); MEK, mitogen-activated protein kinase/ERK kinase; PAQR, progesterone and adipoQ receptor; RKTG, Raf kinase trapping to Golgi; TMD, transmembrane domain.
1To whom correspondence should be addressed (email ychen3@sibs.ac.cn).
Received 13 May 2008/5 June 2008; accepted 11 June 2008
Published as BJ Immediate Publication 11 June 2008, doi:10.1042/BJ20080948
© The Authors Journal compilation © 2008 Biochemical Society
Editorial Advisory Panel
RSS feeds
Table of contents by email
My BJ toolbox
