What should I do with a Gi coupling GPCR?

Hi Le chang, I will do my best to address your question point by point, drawing on my knowledge in this field.

While calcium influx assays can be used for many GPCR targets, it is not a universal method that applies to all GPCRs. The suitability of this method depends on the specific GPCR and its downstream signaling pathways. Some GPCRs may not couple to Gαq/11 proteins, which are necessary for calcium mobilization. Therefore, it's important to assess the signaling pathways and downstream effectors of your specific GPCR of interest before deciding on the assay method.

The theory behind calcium influx assays is based on the fact that the activation of certain GPCRs leads to the activation of Gαq/11 proteins. This activation triggers the release of intracellular calcium stores and the influx of extracellular calcium into the cell. The increase in intracellular calcium levels can be measured using calcium-sensitive dyes or fluorescent indicators, providing a readout of GPCR activity.

If you have tried different ratios of Gα15/Gα16 and GPCR co-transfection without success, there are several other factors you could consider. It's possible that the expression levels of the Gα proteins and the GPCR are not sufficient for robust calcium signaling. Optimizing the expression levels by using different promoters or enhancers may help. Additionally, you could explore different cell lines that are more suitable for GPCR signaling and calcium mobilization.

While I don't have access to specific protocols or examples for a Gi protein-coupled GPCR calcium influx assay, there is a wealth of literature available on GPCR calcium signaling. I recommend searching scientific databases and publications using relevant keywords to find studies that have employed similar assays. Reading through these papers can provide you with valuable insights and protocols. I would like to share an ebook with you. Please check your message inbox as it may be beneficial for your reference.

Adding a Gi-GPCR inhibitor or knocking out Gi proteins may not necessarily increase the chance of GPCR contact with Gα15/16. Gα15/16 proteins are typically involved in GPCR coupling to Gq/11 and not Gi. Modulating Gi signaling might not directly affect the calcium influx mediated by Gα15/16. However, it is important to understand the specific signaling pathways of your GPCR and consider if there are any regulatory mechanisms involving Gi that could impact your calcium influx assay.

There are numerous platforms available where you can discover GPCR discussion forums and interact with experts in the field. Several popular choices include ResearchGate (www.researchgate.net), where you have already asked this question, GPCRdb (www.gpcrdb.org), as well as various scientific forums or communities on social media platforms like Reddit.

Wishing you all the best.

Le Chang

Hi Saif,

Thank you for your excellent and kind answer, which is so helpful. I really appreciate it. One more question, 2. What is the theory of this method? Sorry, I did not make my question clear. What is the theory of this Gi-couple GPCR cotransfect with Gx15/16 method?

Best,

Saif Wahid

Hi Le,

I'm glad to hear that you found my previous answer helpful. Regarding your new question about the Gi-couple GPCR cotransfect with Gx15/16 method, let me provide you with some information.

The Gi-couple GPCR cotransfect with Gx15/16 method is a technique used in molecular biology and pharmacology to study G protein-coupled receptors (GPCRs). GPCRs are a large family of cell surface receptors that are involved in a wide range of physiological processes and are the targets of many drugs.

The theory behind this method is based on the concept of GPCR signaling and the role of G proteins in transmitting signals from GPCRs to intracellular signaling pathways. GPCRs can activate different types of G proteins, including Gs, Gi, and Gq proteins, which initiate various downstream signaling cascades.

In the case of the Gi-couple GPCR cotransfect with Gx15/16 method, the goal is to investigate the signaling pathway mediated by G proteins of the Gi family, specifically Giα-subunits. Giα-subunits are inhibitory G proteins that can inhibit the activity of adenylate cyclase and decrease the production of cyclic AMP (cAMP) in response to GPCR activation.

To study the signaling pathways mediated by Gi-coupled GPCRs, researchers use a transfection approach where both the GPCR of interest (Gi-coupled) and the Gx15/16 protein are introduced into host cells simultaneously. Gx15/16 is an engineered G protein that specifically couples to Gi-coupled GPCRs and enables the downstream signaling cascade.

By cotransfecting the Gi-coupled GPCR and Gx15/16 into host cells, researchers can examine the specific intracellular signaling events triggered by the activation of Gi-coupled GPCRs. This method allows for the investigation of downstream effectors, such as second messenger systems or activation of specific protein kinases or phospholipases, which are involved in the cellular response mediated by Gi-coupled GPCRs.

Overall, the theory behind the Gi-couple GPCR cotransfect with Gx15/16 method revolves around understanding the signaling pathways activated by Gi-coupled GPCRs through the introduction of Gx15/16, which acts as a specific mediator for downstream signaling events.

I hope this explanation clarifies the theory behind the Gi-couple GPCR cotransfect with Gx15/16 method. If you have any further questions, feel free to ask!

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