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Generation of Gαi knock-out HEK293 cells illuminates Gαi-coupling diversity of GPCRs.

ABSTRACT: G-protein-coupled receptors (GPCRs) are pivotal cell membrane proteins that sense extracellular molecules and activate cellular responses. The G-protein α subunit i (Gαi) family represents the most common GPCR-coupling partner and consists of eight subunits with distinct signaling properties. However, analyzing the coupling pattern has been challenging owing to endogenous expression of the Gαi subunits in virtually all cell lines. Here, we generate a HEK293 cell line lacking all Gαi subunits, which enables the measurement of GPCR-Gαi coupling upon transient re-expression of a specific Gαi subunit. We profile Gαi-coupling selectivity across 11 GPCRs by measuring ligand-induced inhibitory activity for cAMP accumulation. The coupling profiles are then classified into three clusters, representing those preferentially coupled to Gαz, those to Gαo, and those with unapparent selectivity. These results indicate that individual Gαi-coupled GPCRs fine-tune Gαi signaling by exerting coupling preference at the Gαi-subunit level.

SUBMITTER: Ono Y

PROVIDER: S-EPMC9884212 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Generation of Gαi knock-out HEK293 cells illuminates Gαi-coupling diversity of GPCRs.

Ono Yuki Y Kawakami Kouki K Nakamura Gaku G Ishida Satoru S Aoki Junken J Inoue Asuka A

Communications biology 20230128 1

G-protein-coupled receptors (GPCRs) are pivotal cell membrane proteins that sense extracellular molecules and activate cellular responses. The G-protein α subunit i (Gαi) family represents the most common GPCR-coupling partner and consists of eight subunits with distinct signaling properties. However, analyzing the coupling pattern has been challenging owing to endogenous expression of the Gαi subunits in virtually all cell lines. Here, we generate a HEK293 cell line lacking all Gαi subunits, wh ...[more]

PMID: 36709222

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Project description:Background: Inactivation of microRNA-100 (miR-100) is involved in hepatocellular carcinoma (HCC) and miR-100 behaves as a tumor suppressor. To understand miR-100 function in HCC genesis and development in vivo, we developed hepatocyte-specific miR-100 deficient mice. Methods: Mice homozygous for floxed miR-100 allele that carried the Alb-Cre transgene (miR-100flox/floxAlb -Cre+) were developed by mating miR-100flox/flox mice with Alb-Cre+/+mice. The mice tails DNA were genotyped using the primers for LoxP sites and Cre recombinase, respectively. The specific deletion of miR-100 in the livers was verified by quantitative Real-time PCR (qRT-PCR). HE-staining was performed for histology analysis. Liver function was assessed by transaminase activity. The metabolic profiles of the hepatocytes were detected using a Seahorse XFe24 extracellular flux analyzer. The direct targets of miR-100 (such as IGF1R-β, mTOR and CDC25A) and HCC related protein (SHP-2) were detected by qRT-PCR and Western blot in liver tissues. Results: The resultant homozygous knockout mice with genotype of miR-100flox/flox-Alb-Cre+ showed an 80% decrease in hepatic miR-100 expression. In adult mice, miR-100 knockout has no effect on the liver function and morphology. In aged mice, HE staining showed that miR-100 knockout caused infiltration of inflammatory cells and expansion of hepatocellular nuclei. Consistently, liver function was impaired in miR-100 knockout aged mice as indicated by increased serum AST and ALT levels. The metabolic analysis demonstrated that the miR-100 knockout hepatocytes tend to adopt glycolysis. The expressions of the miR-100 target genes, such as IGF1R-β, CDC25A and mTOR, were increased. In addition, the known HCC related protein, SHP-2 also was up-regulated in the knockout livers. Conclusions: We successfully generated a miR-100 hepatocyte-specific knock-out mouse model. The malignant transformation related to HCC were observed in aged mice. Therefore, this model is suitable for investigating the mechanism of miR-100 inactivation contributing to HCC genesis in vivo.

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Generation of Gαi knock-out HEK293 cells illuminates Gαi-coupling diversity of GPCRs.

Publications

Generation of Gαi knock-out HEK293 cells illuminates Gαi-coupling diversity of GPCRs.

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