Characterization of the topology and functional domains of RKTG

Abstract

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.

Epithelial-to-mesenchymal transition drives a pro-metastatic Golgi compaction process through scaffolding protein PAQR11

“…On the basis of a prediction algorithm (https://www.predictprotein. org), this N-terminal motif projects into the cytoplasm (Supplemental Figure 10A) and may function as a scaffolding domain similarly to the N-terminus of PAQR3 (33). Protein-binding activity of the PAQR11 N-terminal peptide was greater than that of a control PAQR11 C-terminal peptide (Supplemental Figure 10B).…”

RKTG inhibits angiogenesis by suppressing MAPK-mediated autocrine VEGF signaling and is downregulated in clear-cell renal cell carcinoma

“…RKTG (Raf Kinase Trapping to Golgi), a member of the PAQR (Progestin and AdipoQ Receptors) family, is a seven-transmembrane protein specifically located at the Golgi apparatus in mammalian cells (Feng et al, 2007;Luo et al, 2008). Our previous studies have revealed that RKTG acts as a spatial regulator of Raf kinase by sequestering Raf to the Golgi apparatus, thereby antagonizing the mitogen-activated protein kinase (Raf/MEK/ERK) signaling cascade (Feng et al, 2007;Fan et al, 2008).…”

Nutrient-dependent phosphorylation channels lipid synthesis to regulate PPARα

“…Solubility assays were performed as previously described ( 15 ) with minor modifi cations. Membranes were isolated from mouse liver by ultracentrifugation and resuspended in buffer containing 20 mM HEPES buffer (pH 7.4), 1 mM EDTA, and 255 mM sucrose.…”