Mini G Protein Inhibits Internalization of Same GPCR and Disrupts Downstream Intracellular Signaling

Introduction

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, regulating cellular responses to external stimuli such as hormones and neurotransmitters. GPCRs transmit signals by coupling with guanine nucleotide-binding regulatory proteins (G proteins). The binding of an agonist induces a conformational change in the receptor, which then activates the trimeric G protein complex. This change initiates a signaling cascade that can lead to specific cellular effects. After signal transmission, the inherent GTPase activity of the Gα subunit returns it to its inactive GDP-bound state.

In recent years, to better study the structure of GPCRs, scientists have adopted the strategy of co-expressing corresponding thermostable Gα subunit mini G proteins. These mini G proteins can stabilize the active conformation of GPCRs. However, as the use of mini G proteins becomes more widespread, it is necessary to carefully define the potential effects of mini G co-expression on GPCR internalization and intracellular signaling.

Research Source

This paper is authored by Yusman Manchanda et al., with authors from Imperial College London, Tohoku University, and Université de Montréal, among others. The article was published in the journal Science Signaling on July 2, 2024.

Experimental Methods

The authors reported that overexpression of mini G proteins with their corresponding GPCRs leads to impaired GPCR internalization and disruption of related intracellular signaling. To investigate this phenomenon in detail, the authors conducted the following experimental steps:

Experimental Procedures

1. Co-expression of GPCR and Mini Gs:

   • In HEK293 cells expressing the Gαs-coupled GPCR glucagon-like peptide-1 receptor (GLP-1R), mini Gs and GLP-1R were co-expressed. Fluorescent or luminescent tags were used to label mini Gs and GLP-1R to observe their distribution and internalization in cells.

2. NanoBRET (Nano Bioluminescence Resonance Energy Transfer) Experiment:

   • Using NanoBRET technology, the energy transfer at different time points was measured in HEK293 cells co-expressing mini Gs and GLP-1R to monitor mini Gs membrane residence, internalization, and intracellular signaling.

3. Fluorescence Colocalization Microscopy Analysis:

   • Stepwise colocalization techniques were used in HEK293 and INS-1 ⁸³²⁄₃ cells to observe the colocalization of mini Gs and GLP-1R with the early endosome marker Rab5.

4. β-arrestin and GRK2 Recruitment:

   • Using NanoBRET and NanoBiT technologies, the recruitment of β-arrestin 2 and GRK2 (GPCR kinase 2) was detected during mini Gs co-expression:

     • Fluorescent tags were used to measure energy transfer in different conditions on GLP-1R, monitoring the recruitment of β-arrestin 2 and GRK2 under various conditions.

Research Results

1. Mini Gs Inhibits GLP-1R Internalization:

   • Main Finding One: In cells co-expressing mini Gs, GLP-1R does not effectively internalize in response to agonist stimulation.
   • Supporting Data: NanoBRET experiments and colocalization microscopy analysis showed that the NanoBRET signal did not diminish with GLP-1R internalization over time, indicating that mini Gs remained at the cell membrane.

2. Mini G Subunits Selectively Inhibit Corresponding GPCR Internalization:

   • Co-expression of different types of mini G proteins (mini Gi and mini Gq) was also able to inhibit internalization of their respective GPCRs.
   • Co-expression of mini G subunits did not effectively inhibit the action of endogenous Gα, indicating that full-length Gα subunits had a smaller impact on GPCR internalization compared to mini G proteins.

3. β-arrestin 2 Recruitment Is Affected:

   • Complete inhibition of β-arrestin 2 recruitment suggests that β-arrestin 2 recruitment requires the dissociation of the Gα subunit, whereas GRK2 recruitment was only slightly affected and did not significantly alter GLP-1R phosphorylation status.

4. Effect of Mini G Proteins on GPCR-Ligand Binding Affinity:

   • In cells co-expressing mini Gs, the ligand binding affinity of GLP-1R increased, indicating that the receptor remained in a stable active conformation.

Conclusion and Significance

This study is the first to reveal the impact of mini G protein co-expression on the internalization and downstream signaling of their corresponding GPCRs. One of the key findings is that mini G proteins can prevent GPCR internalization, thereby altering their intracellular signaling properties. Additionally, this study has implications for designing new methods for assessing GPCR signaling, guiding future experiments to avoid misconceptions about mini G subunit overexpression, and enhancing the accuracy of GPCR molecular pharmacology and signaling research.

Research Highlights and Innovations

The selective internalization inhibition of different mini G subunits upon mini G protein co-expression is reported for the first time. This discovery is significant not only in academic research but also in promoting the discovery of new drug targets and the development of therapies. Furthermore, the paper proposes a new method using nanobody Nb37, which can more accurately detect intracellular signal transduction without affecting GLP-1R internalization.

Summary

This paper reveals the significant inhibitory effect of mini G proteins on the internalization and downstream signaling of their corresponding GPCRs, providing new perspectives and methods for future research on GPCR signaling. Through these in-depth experiments and observations, this paper provides valuable data for the scientific community, deepening the understanding of the mechanisms of GPCR signal transduction.