Caveolin-1 prevents palmitate-induced NF-κB signaling by inhibiting GPRC5B-phosphorylation

https://doi.org/10.1016/j.bbrc.2018.08.022Get rights and content

Abstract

Tyrosine phosphorylation of GPRC5B and phosphorylation-dependent recruitment of Fyn through the SH2 domain have been implicated in NF-κB activation and obesity-linked adipose inflammation. GPRC5B tightly associates with caveolin-1 (Cav1); however, the role of this interaction remains elusive. Here, we report that Cav1 reduces GPRC5B-mediated NF-κB signaling by blocking GPRC5B-phosphorylation. We demonstrate highly abundant tyrosine phosphorylation of GPRC5B is observed in Neuro2a cells lacking endogenous Cav1 expression. Reversely, exogenous expression of Cav1 in these cells inhibits GPRC5B-phosphorylation. Although GPRC5B lacks conventional caveolin-binding motif, cytoplasmic tail of GPRC5B directly interacts with the C-terminal domain of Cav1. The vacant scaffolding domain of Cav1 in the protein complex suggests a potential mechanism for blocking GPRC5B-phosphorylation by Cav1, because Fyn loses the activity by binding with Cav1-scaffolding domain. Enhanced GPRC5B-mediated NF-κB signaling in Cav1-deficient cells were observed under palmitate-induced metabolic stress. These results support Cav1 functions as a negative modulator for GPRC5B action.

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).

References (23)

  • R.G. Baker et al.

    NF-κB, inflammation and metabolic disease

    Cell Metabol.

    (2011)
  • Cited by (10)

    • The orphan receptor GPRC5B modulates inflammatory and fibrotic pathways in cardiac fibroblasts and mice hearts

      2019, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      Additionally, Kim et al. showed that GPRC5B-deficiency protects mice from diet-induced obesity and insulin resistance through a reduction of chronic inflammation in white adipose tissue. Interestingly, GPRC5B has been shown to act through the NFκB-pathway by stimulating the kinase-activity of Fyn which can be inhibited by Caveolin-1 [7,8]. A study by Atanes et al. using β-cells suggested a regulatory role of GPRC5B in TGF-β- and IFN-γ-signaling which are pathways that are strongly involved in fibrosis [9] and inflammation [10], respectively [11].

    View all citing articles on Scopus
    View full text