Studying G protein-coupled receptors: immunoblotting, immunoprecipitation, phosphorylation, surface labeling, and cross-linking protocols

Pal, Kasturi; Badgandi, Hemant B.; Mukhopadhyay, Sutapa

doi:10.1016/bs.mcb.2014.12.003

Cited by 10 publications

(14 citation statements)

References 37 publications

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“…The resulting immunoprecipitates were western blotted with indicated antibodies. We note that immunoblotting data indicate multiple bands for GPCRs (indicated by the square brackets on the right of gel images) probably because GPCRs tend to aggregate after boiling in Laemmli buffer (Pal et al, 2015).…”

Section: Discussionmentioning

confidence: 98%

RABL2 positively controls localization of GPCRs in mammalian primary cilia

Dateyama

Sugihara

Chiba

et al. 2019

Journal of Cell Science

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The primary cilium, a solitary protrusion from most mammalian cells, functions as a cell sensor by receiving extracellular signals through receptors and channels accumulated in the organelle. Certain G-protein-coupled receptors (GPCRs) specifically localize to the membrane compartment of primary cilia. To gain insight into the mechanisms that regulate ciliary GPCR sorting, we investigated the atypical small GTPase RAB-like 2 (RABL2; herein referring to the near-identical human paralogs RABL2A and RABL2B). RABL2 recruitment to the mother centriole is dependent on the distal appendage proteins CEP164 and CEP83. We found that silencing of RABL2 causes mis-targeting of ciliary GPCRs, GPR161 and HTR6, whereas overexpression of RABL2 resulted in accumulation of these receptors in the organelle. Ablation of CEP19 and the intraflagellar transport B (IFT-B) complex, which interact with RABL2, also leads to mis-localization of GPR161. RABL2 controls localization of GPR161 independently of TULP3, which promotes entry of ciliary GPCRs. We further demonstrate that RABL2 physically associates with ciliary GPCRs. Taken together, these studies suggest that RABL2 plays an important role in trafficking of ciliary GPCRs at the ciliary base in mammalian cells.

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Section: Discussionmentioning

confidence: 98%

RABL2 positively controls localization of GPCRs in mammalian primary cilia

Dateyama

Sugihara

Chiba

et al. 2019

Journal of Cell Science

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“…Methods for GPCR immunoblotting have been described elsewhere ( Pal et al, 2015 ). In brief, cells were harvested by scraping in cold TBS (150 mM NaCl, 50 mM Tris, pH 7.5, 2 mM EDTA, and 1 mM EGTA), lysed in the same buffer supplemented with 1% digitonin, and protease/phosphatase inhibitors, clarified at 16,000 g for 10 min, assessed for protein levels and subsequently treated with 2× urea sample buffer (4 M urea, 4% SDS, 100 mM Tris, pH 6.8, 0.2% bromophenol blue, 20% glycerol, and 200 mM DTT) at room temperature for 1 h. Equal amounts of protein were loaded to 4–15% Mini-PROTEAN TGX precast gels (Bio-Rad Laboratories, Inc.) and immunoblotted for GFP and α-tubulin.…”

Section: Methodsmentioning

confidence: 99%

Smoothened determines β-arrestin–mediated removal of the G protein–coupled receptor Gpr161 from the primary cilium

Pal

Hwang

Somatilaka

et al. 2016

Journal of Cell Biology

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159

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“…The cells were lysed with digitonin lysis buffer as described (38). The proteins were immunoblotted with anti-HA (Santa Cruz Biotechnology, Dallas, TX), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling, Danvers, MA), 1RDye ® 800CW goat anti-rabbit IgG secondary antibodies (LI-COR, Cambridge, UK) and 1RDye ® 680CW goat anti-mouse IgG secondary antibodies (LI-COR).…”

Section: Western Blot Analysismentioning

confidence: 99%

Dominant negative GPR161 rare variants are risk factors of human spina bifida

Kim

Lei

Hwang

et al. 2018

Human Molecular Genetics

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Spina bifida (SB) is a complex disorder of failed neural tube closure during the first month of human gestation, with a suspected etiology involving multiple gene and environmental interactions. GPR161 is a ciliary G-protein coupled receptor that regulates Sonic Hedgehog (Shh) signaling. Gpr161 null and hypomorphic mutations cause neural tube defects (NTDs) in mouse models. Herein we show that several genes involved in Shh and Wnt signaling were differentially expressed in the Gpr161 null embryos using RNA-seq analysis. To determine whether there exists an association between GPR161 and SB in humans, we performed direct Sanger sequencing on the GPR161 gene in a cohort of 384 SB patients and 190 healthy controls. We identified six rare variants of GPR161 in six SB cases, of which two of the variants were novel and did not exist in any databases. Both of these variants were predicted to be damaging by SIFT and/or PolyPhen analysis. The novel GPR161 rare variants mislocalized to the primary cilia, dysregulated Shh and Wnt signaling and inhibited cell proliferation in vitro. Our results demonstrate that GPR161 mutations cause NTDs via dysregulation of Shh and Wnt signaling in mice, and novel rare variants of GPR161 can be risk factors for SB in humans.

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Studying G protein-coupled receptors: immunoblotting, immunoprecipitation, phosphorylation, surface labeling, and cross-linking protocols

Cited by 10 publications

References 37 publications

RABL2 positively controls localization of GPCRs in mammalian primary cilia

RABL2 positively controls localization of GPCRs in mammalian primary cilia

Smoothened determines β-arrestin–mediated removal of the G protein–coupled receptor Gpr161 from the primary cilium

Dominant negative GPR161 rare variants are risk factors of human spina bifida

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