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Regulation of M? muscarinic receptor expression and function by transmembrane protein 147.

ABSTRACT: The M? muscarinic acetylcholine receptor (M3R) regulates many fundamental physiological functions. To identify novel M3R-interacting proteins, we used a recently developed yeast two-hybrid screen (split ubiquitin method) to detect interactions among membrane proteins. This screen led to the identification of many novel M3R-associated proteins, including the putative membrane protein transmembrane protein 147 (Tmem147). The amino acid sequence of Tmem147 is highly conserved among mammals, but its physiological roles are unknown at present. We initially demonstrated that Tmem147 could be coimmunoprecipitated with M3Rs in cotransfected mammalian cells (COS-7 cells). Confocal imaging studies showed that Tmem147 was localized to endoplasmic reticulum (ER) membranes and that the Tmem147/M3R interaction occurred in the ER of cotransfected COS-7 cells, resulting in impaired trafficking of the M3R to the cell surface. To study the role of Tmem147 in modulating M3R function in a more physiologically relevant setting, we carried out studies with H508 human colon cancer cells that endogenously express M3Rs and Tmem147. Treatment of H508 cells with carbachol, a hydrolytically stable acetylcholine analog, promoted H508 cell proliferation and activation of the mitogenic kinase, p90RSK. Small interfering RNA-mediated knockdown of Tmem147 expression significantly augmented the stimulatory effects of carbachol on H508 cell proliferation and p90RSK activation. These effects were associated with an increase in the density of cell surface M3Rs. Our data clearly indicate that Tmem147 represents a potent negative regulator of M3R function, most likely by interacting with M3Rs in an intracellular compartment (ER). These findings may lead to new strategies aimed at modulating M3R activity for therapeutic purposes.

SUBMITTER: Rosemond E

PROVIDER: S-EPMC3033710 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Regulation of M₃ muscarinic receptor expression and function by transmembrane protein 147.

Rosemond Erica E Rossi Mario M McMillin Sara M SM Scarselli Marco M Donaldson Julie G JG Wess Jürgen J

Molecular pharmacology 20101105 2

The M₃ muscarinic acetylcholine receptor (M3R) regulates many fundamental physiological functions. To identify novel M3R-interacting proteins, we used a recently developed yeast two-hybrid screen (split ubiquitin method) to detect interactions among membrane proteins. This screen led to the identification of many novel M3R-associated proteins, including the putative membrane protein transmembrane protein 147 (Tmem147). The amino acid sequence of Tmem147 is highly conserved among mammals, but its ...[more]

PMID: 21056967

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Project description:BackgroundRecombinant galectins of male and female Haemonchus contortus (rHco-gal-m/f) have been recognized as significant regulators of the functions of goat peripheral blood mononuclear cells (PBMC). In previous research, transmembrane protein 63A (TMEM63A) was identified as a partner protein in the regulation associated with H. contortus infection. However, in the identification of binding partners for galectins of male and female H. contortus (Hco-gal-m/f) by yeast two-hybrid (YTH) screening, it was found that the transmembrane protein 147 (TMEM147) could also bind to Hco-gal-m/f. In this study, the functions of TMEM147 in the regulations of H. contortus galectin on the goat PBMC were investigated.MethodsTo identify Hco-gal-m/f-interacting proteins, a yeast two-hybrid system to detect interactions was used. Co-immunoprecipitation and immunoblotting were used to validate the interaction between recombinant galectins of male H. contortus (rHco-gal-m) and candidate binding protein. The localization of TMEM147 in PBMC was explored by immunofluorescence in confocal imaging studies. Flow cytometry was used to determine the distribution of TMEM147 in T cells, B cells and monocytes in PBMC. The modulatory effects of rHco-gal-m and TMEM147 on cell proliferation, phagocytosis, nitric oxide production, migration, apoptosis and cytokine mRNA transcription were observed by co-incubation of rHco-gal-m and knockdown of the tmem147 gene.ResultsIn this research, it was demonstrated that TMEM147 could bind to rHco-gal-m/f. Immunofluorescence assays showed that TMEM147 was localized to the cell membrane and within the cell membrane in goat PBMC. Flow cytometric analysis revealed that TMEM147 was expressed in all B cells and monocytes in goat PBMC. However, 3.8 % of T cells did not express this protein. Knockdown of the tmem147 gene using RNA interference (RNAi) showed that the interaction of galectin with TMEM147 mainly mediated cell proliferation, cell apoptosis, transcription of interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) of goat PBMC. This membrane protein, together with TMEM63A, was also related to the regulation of galectin on phagocytosis and nitric oxide production of goat PBMC. However, it might not be involved in the regulation of galectin on the migration and interferon-γ (IFN-γ) transcription of goat PBMC.ConclusionsOur results showed that TMEM147 was a binding partner of Hco-gal-m/f and mediated the immunological regulation of Hco-gal-m/f on goat PBMC in a manner different to that of TMEM63A.

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Regulation of M? muscarinic receptor expression and function by transmembrane protein 147.

Publications

Regulation of M₃ muscarinic receptor expression and function by transmembrane protein 147.

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