Biochemical and Biophysical Research Communications
Caveolin-1 prevents palmitate-induced NF-κB signaling by inhibiting GPRC5B-phosphorylation
Introduction
GPRC5B was initially identified as a retinoic acid-induced gene product [1]. It belongs to GPRC5 family that is comprised of GPRC5A, GPRC5B, GPRC5C and GPRC5D in mammals, and its amino acid sequence is similar to G protein-coupled receptors (GPCR) in the class C family, such as metabotropic glutamate receptors, γ-aminobutyric acid receptors, and taste receptors. Although it has a similar sequence signature for GPCR, its endogenous agonist and G protein-related signaling are completely unknown.
A genome-wide sequence analysis revealed a strong correlation between body mass index and the presence of a 21-kb copy number variation upstream of the human GPRC5B gene suggesting its crucial role in metabolic regulation [2]. And the GPRC5B gene is evolutionarily conserved in Drosophila, and its orthologue in fly is called BOSS. BOSS-deficient fly shows abnormal energy metabolism, insulin signaling, change of feeding behavior, and shortened lifespan [[3], [4], [5]]. GPRC5B deficiency in mice [6] protects from diet-induced obesity and insulin resistance the underlying mechanism by which GPRC5B recruits Fyn involved in NF-κB activation implicated in chronic inflammation in adipose tissues [7]. These suggest that GPRC5B plays a significant biological role in the metabolic regulation of insulin-sensitive organs, including the central nervous system, muscles, and adipose tissues.
Cav1 is an integral membrane protein that has multi-functional features, such as organization of membrane rafts/caveolae and scaffolding various factors to regulate cellular signaling cascades [8]. Our previous study showed GPRC5B was localized in membrane rafts, and tightly associated with Cav1 [7]. However, the role of Cav1 affects to GPRC5B has not been investigated. Therefore, we focus on the potential role of Cav1 in GPRC5B-derived signaling, in particular, metabolic stress-induced NF-κB activation.
Section snippets
Antibodies and plasmids
Anti-GPRC5B rabbit polyclonal antibody and anti-phosphosite-specific antibodies were described previously [7]. Anti-phosphotyrosine (P-Tyr-1000), anti-caveolin-1, anti-IκBα (L35A5), anti-a-tubulin (DM1A) and anti-flotillin-1 antibodies were purchased from Cell Signaling Technology. Anti-transferrin receptor antibody was purchased from Thermo Fisher Scientific. Anti-GFP antibody (GF200) was purchased from Nacalai tesque. Expression plasmids encoding GPRC5B-3 × Flag and GPRC5B-AcGFP were
GPRC5B interacts with Cav1 in the plasma membrane
GPRC5B is mainly localized in membrane rafts [7]. Membrane rafts are heterogeneous, cholesterol and sphingomyelin enriched membrane domains which are considered as critical compartment for regulating cellular signaling events [11]. To confirm the subcellular localization of GPRC5B, we performed immunofluorescence confocal microscopy in GPRC5B-AcGFP-transfected HEK293 cells. Endogenous Cav1 showed punctate cell surface staining merged with GPRC5B-AcGFP in the plasma membrane (Fig. 1A),
Discussion
Cav1 is a major component of caveolae which are flask shaped invaginations of the plasma membrane regulating a number of signaling cascades, endocytosis, and cholesterol homeostasis [16]. This unique specialized lipid rafts structure may have a significant biological role maintaining cellular homeostasis, since a number of mutations in Cav1 and Cav3 gene have been reported in breast cancer [17] and caveolinopathies [18], respectively. In addition, caveolins and caveolae are also involved in
Acknowledgements
We thank professor J. Inokuchi for kindly providing Cav1 knockout MEFs. We thank MS. Y. Asano for technical assistance. We are grateful to the Support Unit for Bio-Material Analysis, Research Resources Division, RIKEN Center for Brain Science, for help with the nucleotide sequencing analysis. This work was in part supported by Integrated Lipidology Program of RIKEN (YH), and RIKEN Center for Brain Science (YH).
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