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Gαs Inhibitory Probes Kits
(Kit # 1000000287, 1000000288, 1000000289 )

Depositing Lab:   Mikel Garcia-Marcos

Kit 1000000287: The Gαs inhibitory probes (pcDNA3.1) kit contains plasmids in a pcDNA3.1 backbone for the expression of constructs containing the Gαs binding peptide 2 (GαsBP2) sequence.

Kit 1000000288: The Gαs inhibitory probes (chemogenetically inducible inhibition) kit contains plasmids for the expression of constructs to achieve chemically-induced inhibition of Gαs by two different approaches: one based on rapamycin and another one based on the plant pheromone abscisic acid (ABA).

Kit 1000000289: The Gαs inhibitory probes (lentiviral constructs) kit contains plasmids in a pLVX backbone compatible with lentiviral packaging for the expression of constructs containing the Gαs binding peptide 2 (GαsBP2) sequence.

These kits will be sent as individual bacterial stabs at room temperature.

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Ordering

Item Catalog # Description Quantity Price (USD)
Kit 1000000287 Gαs Inhibitory Probes (pcDNA3.1) Kit 1 $423 Add to Cart
Kit 1000000288 Gαs Inhibitory Probes (chemogenetically inducible inhibition) Kit 1 $423 Add to Cart
Kit 1000000289 Gαs Inhibitory Probes (lentiviral constructs) Kit 1 $423 Add to Cart
This material is available to academics and nonprofits only.

Original Publication

Inhibitory probes for spatiotemporal analysis of Gαs protein signaling. Zhao J, Luebbers A, Savransky S, Lin TY, Cheng N, Wilcox A, Janicot R, Green E, Sharma A, Maziarz M, Varelas X, Irannejad R, Vilardaga JP, Garcia-Marcos M. Nat Chem Biol. 2026 Feb 3. doi: 10.1038/s41589-025-02138-1. PubMed (Link opens in a new window) Article (Link opens in a new window)

Description

Kit 1000000287: The Gαs inhibitory probes (pcDNA3.1) kit contains plasmids in a pcDNA3.1 backbone for the expression of constructs containing the Gαs binding peptide 2 (GαsBP2) sequence. These constructs serve as genetically encoded probes that specifically bind the active conformation of Gαs and block its ability to interact with effectors like adenylyl cyclase, thereby preventing G protein signal propagation (Figure 1). The different variants included in this kit include probes that can be targeted to specific subcellular compartments (plasma membrane, endosomes, cytosol) and negative controls consisting of a mutant (W11A) that does not bind to Gαs. All constructs are fusions to the red fluorescent protein mKate2. Together, these constructs provide a versatile platform for studying the spatiotemporal organization of endogenous Gαs signaling.

Kit 1000000288: The Gαs inhibitory probes (chemogenetically inducible inhibition) kit contains plasmids for the expression of constructs to achieve chemically-induced inhibition of Gαs by two different approaches: one based on rapamycin and another one based on the plant hormone abscisic acid (ABA) (Figure 2). Each approach consists of a chemically induced dimerization system of two components expressed in separate plasmids. One component contains the Gαs binding peptide 2 (GαsBP2) sequence, which serves as a genetically encoded probe that specifically binds the active conformation of Gαs and blocks its ability to interact with effectors like adenylyl cyclase. This component is fused to one of two dimerization domains. Upon rapamycin or ABA stimulation, this domain dimerizes with a second domain that is targeted to the plasma membrane and expressed from a separate plasmid. As a consequence, the GαsBP2-containing construct is translocated from the cytosol to the plasma membrane. GαsBP2 is inactive in the cytosol. Once translocated to the plasma membrane, it becomes competent to inhibit Gαs signaling efficiently.

The rapamycin-inducible system is based on the FKBP-FRB pair of domains, whereas the ABA-inducible system is based on the ABIcs–PYLcs pair of domains.

Kit 1000000289: The Gαs inhibitory probes (lentiviral constructs) kit contains plasmids in a pLVX backbone compatible with lentiviral packaging for the expression of constructs containing the Gαs binding peptide 2 (GαsBP2) sequence. These constructs serve as genetically encoded probes that specifically bind the active conformation of Gαs and block its ability to interact with effectors like adenylyl cyclase, thereby preventing G protein signal propagation (Figure 1). The different construct variants included in this kit include probes that can be targeted to specific subcellular compartments (plasma membrane, endosomes, cytosol) and negative controls consisting of a mutant (W11A) that does not bind to Gαs. All constructs are fusions to the red fluorescent protein mKate2. Together, these constructs provide a versatile platform for studying the spatiotemporal organization of endogenous Gαs signaling.

Structure of Gαs binding peptide 2 (GαsBP2) binding the α3 helix and SwII of Gαs. To the right a schematic of Gαs signaling modulation shows the plasma membrane with transmembrane protein GPCR binding its ligand. This leads to active Gαs. When GαsBP2 inhibits active Gαs, AC leads to cAMP. To the right are three additional schematics of mem-GαsBP2, endo-GαsBP2, and cyto-GαsBP2. The mem-GαsBP2 inhibits Gαs at the membrane but not in the endosome. The endo-GαsBP2 inhibits Gαs in the endosome but not the plasma membrane. The Cyto-GαsBP2 inhibits Gαs at the membrane and in the endosome.

Figure 1: Gαs binding peptide 2 (GαsBP2) is a genetically encoded peptide that specifically binds active Gαs and blocks its interaction with adenylyl cyclase (AC). GαsBP2 specifically targeted to subcellular loci enable the precise modulation of endogenous Gαs signaling within defined cellular compartments.

Rapamycin induced Gαs signaling inhibition and Abscisic acid (ABA) induced Gαs signaling inhibition. For rapamycin induced Gαs signaling inhibition, an insert shows when the ligand rapamycin is added in the presence of FRB and FKBP, they bind together. In the presence of the ligand isoproterenol, the transmembrane protein beta2AR activates Gαs/GTP which is bound to the transmembrane protein Adenylyl cyclase (AC). FRB is tethered to the cytosolic side of the plasmids membrane and with FKBP-bound GαsBP2 and in the presence of rapamycin it inhibits Gαs/GTP. Abscisic acid (ABA) induced Gαs signaling inhibition works exactly the same when the ligand ABA is added in the presence of ABIcs and PYLcs.

Figure 2: Under resting conditions, GαsBP2 constructs in the cytosol do not affect Gαs signaling. Rapamycin- or ABA-induced recruitment of GαsBP2 from the cytosol to membranes leads to efficient and specific inhibition of Gαs signaling.

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:

"The Gαs Inhibitory Probes (pcDNA3.1) Kit was a gift from Mikel Garcia-Marcos (Addgene kit #1000000287)."
"The Gαs Inhibitory Probes (Chemogenetically Inducible Inhibition) Kit was a gift from Mikel Garcia-Marcos (Addgene kit #1000000288)."
"The Gαs Inhibitory Probes (lentiviral constructs) Kit was a gift from Mikel Garcia-Marcos (Addgene kit #1000000289)."

For your Reference section:

Inhibitory probes for spatiotemporal analysis of Gαs protein signaling. Zhao J, Luebbers A, Savransky S, Lin TY, Cheng N, Wilcox A, Janicot R, Green E, Sharma A, Maziarz M, Varelas X, Irannejad R, Vilardaga JP, Garcia-Marcos M. Nat Chem Biol. 2026 Feb 3. doi: 10.1038/s41589-025-02138-1. PubMed (Link opens in a new window) Article (Link opens in a new window)

The Gαs inhibitory probes (pcDNA3.1) kit contains six plasmids. Please refer to the individual plasmid pages below for more details on each plasmid in this kit:

The Gαs inhibitory probes (chemogenetically inducible inhibition) kit contains four plasmids. Please refer to the individual plasmid pages below for more details on each plasmid in this kit:

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