Gbetagamma subunits of heterotrimeric G proteins. GRK2i TFA is a polypeptide inhibitor derived from the Gbetagamma-binding domain of G protein-coupled receptor kinase 2 (GRK2). GRK2 (also known as beta-adrenergic receptor kinase 1, ADRBK1) is a serine/threonine kinase that phosphorylates and desensitizes agonist-activated GPCRs. GRK2 is activated by binding to Gbetagamma dimers, which translocate the kinase to the plasma membrane. GRK2i competes with endogenous GRK2 for Gbetagamma binding, acting as a cellular antagonist. By binding to Gbetagamma, GRK2i prevents Gbetagamma from activating GRK2 and also blocks Gbetagamma-mediated signaling pathways, such as activation of PI3Kgamma, PLCbeta, and certain ion channels, independent of GRK2. Thus, GRK2i is a broad inhibitor of Gbetagamma-mediated effects. The peptide is not selective for GRK2 over other Gbetagamma effectors, as it directly targets the Gbetagamma subunit.

In vitro, GRK2i TFA (a 29-amino acid polypeptide) binds specifically to Gbetagamma protein dimers, preventing their interaction with GRK2 and other Gbetagamma effectors. The peptide's inhibitory activity has been demonstrated in reconstituted phospholipid vesicle assays, where it blocks Gbetagamma-mediated activation of GRK2 and other effectors such as PI3Kgamma and adenylyl cyclase. At concentrations of 1-50 uM, GRK2i inhibits Gbetagamma-stimulated GRK2 activity and reduces Gbetagamma-dependent signaling events in cell-free systems. The peptide (10-100 uM) can also block Gbetagamma-induced activation of PLCbeta and GIRK channels in reconstituted systems. The inhibition is competitive with respect to Gbetagamma binding, as the peptide contains the Gbetagamma-binding motif from GRK2. The IC50 in cell-free assays for inhibiting Gbetagamma-GRK2 interaction is in the 0.5-5 uM range, depending on assay conditions. The peptide is also used as a molecular tool to block Gbetagamma signaling in permeabilized cell assays. No direct receptor binding is involved; the target is the Gbetagamma subunit of heterotrimeric G proteins. The TFA salt form does not affect peptide activity.

In vivo, GRK2i TFA has been used in cell culture and ex vivo experiments to block Gbetagamma signaling in various systems. For example, in neuronal cultures (e.g., primary hippocampal or cortical neurons), GRK2i (10-50 uM) inhibits Gbetagamma-mediated neurite retraction, cell migration, and axon guidance. In cardiac myocytes, GRK2i (10-50 uM, delivered by microinjection or via cell-permeable peptide variants) blocks Gbetagamma-dependent signaling pathways, including PI3Kgamma and MAPK activation, and reduces contractile responses. In ex vivo tissue preparations (e.g., isolated heart or brain slices), GRK2i peptide (1-20 uM) is typically applied via superfusion. However, because GRK2i is a large hydrophilic peptide (29 amino acids, MW ~3.6 kDa), it does not readily cross cell membranes; therefore, in vivo studies require delivery strategies such as microinjection, conjugation to cell-penetrating peptides (CPPs) (e.g., TAT-GRK2i), or viral delivery (e.g., adeno-associated virus, AAV). Non-modified GRK2i is not suitable for systemic in vivo administration. The peptide is widely used as a research tool to validate the role of Gbetagamma signaling in physiological and pathological contexts, including cardiac hypertrophy, inflammation, and neurological disorders.

For non-cellular binding assays, a direct binding between GRK2i and Gbetagamma can be measured using surface plasmon resonance (SPR) or fluorescence polarization (FP). For SPR: Recombinant Gbetagamma subunits (e.g., Gbeta1gamma2) are immobilized on a CM5 sensor chip via amine coupling (EDC/NHS chemistry) or captured using an anti-Gbeta antibody. GRK2i TFA is dissolved in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20, 1 mM DTT). Increasing concentrations of GRK2i (0.001-100 uM) are flowed over the immobilized Gbetagamma at 25degC with a flow rate of 30 uL/min. Association (2 min) and dissociation (5 min) phases are recorded. The sensorgrams are double-referenced. The KD (dissociation constant) is calculated by fitting to a 1:1 Langmuir binding model. For FP: Fluorescein-labeled GRK2i (FITC-GRK2i) is prepared. In a 384-well black plate, a fixed concentration of FITC-GRK2i (10-50 nM) is incubated with varying concentrations of unlabeled GRK2i (0.01-100 uM) in the presence of Gbetagamma (10-50 nM) in binding buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.01% NP-40). After equilibrium (30-60 min at room temperature), fluorescence polarization is measured (excitation 485 nm, emission 520 nm). The IC50 for unlabeled GRK2i is determined, and the Ki is calculated. Alternatively, a competitive ELISA can be used: Immobilize Gbetagamma on a 96-well plate, add biotinylated GRK2i (biotin-GRK2i) in the absence or presence of unlabeled GRK2i, detect with streptavidin-HRP, and measure absorbance at 450 nm. For a Gbetagamma-GRK2 interaction assay using pull-down: Recombinant GRK2 (or GRK2 PH domain) is incubated with Gbetagamma and GRK2i, then immunoprecipitated with anti-GRK2 antibody, and Gbetagamma detected by Western blot.

For cell-based assays, cells (e.g., HEK293, COS-7, primary neurons, or cardiac myocytes) are used. However, the GRK2i peptide is not cell-permeable due to its large size and hydrophilicity; thus, it must be delivered by microinjection, electroporation, or conjugation to a cell-penetrating peptide (CPP) (e.g., TAT, penetratin, or Antp). For studies using microinjection: Cells are plated on glass coverslips and microinjected with GRK2i TFA (1-100 uM in injection buffer) using an Eppendorf FemtoJet or similar micromanipulator. For CPP-conjugated GRK2i: TAT-GRK2i (where TAT peptide YGRKKRRQRRR is fused to GRK2i) or myristoylated GRK2i (myr-GRK2i) is commercially available. Cells are treated with CPP-GRK2i (1-50 uM) in serum-free medium for 2-6 hours at 37degC. For a typical Gbetagamma signaling assay: Cells expressing a GPCR (e.g., chemokine receptor CXCR4, angiotensin II AT1R, or beta2-adrenergic receptor) are serum-starved overnight. Then, cells are pretreated with CPP-GRK2i (10-50 uM) for 2-4 hours, followed by agonist stimulation (e.g., SDF-1alpha/CXCL12, angiotensin II, or isoproterenol) for 5-30 minutes. Downstream signaling is assessed by Western blot for phosphorylated ERK1/2 (p-ERK, indicating MAPK activation), phosphorylated AKT (p-AKT, indicating PI3K activation), or other relevant readouts. Alternatively, a transcription factor reporter assay (e.g., NF-kappaB-luciferase or SRE-luciferase) can be used. Inhibition of Gbetagamma signaling by GRK2i is indicated by reduced phosphorylation or luciferase activity. For functional assays (e.g., cell migration): Use a Transwell migration assay. Cells are treated with CPP-GRK2i (10-50 uM) for 2-4 hours, then seeded into the upper chamber of Transwell inserts (8-um pores) with serum-free medium. The lower chamber contains chemoattractant (e.g., CXCL12, 10-100 ng/mL). After 4-24 hours, migrated cells on the lower surface are stained with crystal violet or Calcein-AM, and quantified. GRK2i is expected to inhibit chemokine-induced migration. All experiments should include a scrambled GRK2i peptide as a negative control. For GIRK channel assays: In CHO or HEK cells co-expressing GIRK1/4 channels and a GPCR (e.g., GABAB or adenosine A1 receptor), whole-cell patch-clamp electrophysiology can be performed. GRK2i (5-50 uM, via internal pipette solution) is dialyzed into the cell, and GIRK current inhibition is measured. Each condition should be performed in triplicate wells or cells, with at least 3 independent experiments. GRK2i TFA stock solutions: Dissolve in sterile water or PBS at 1-10 mM, store at -20degC, avoid freeze-thaw cycles.

For in vivo studies, the naked GRK2i TFA is not used because it does not cross cell membranes and has a short plasma half-life. To study GRK2i in vivo, researchers typically use a cell-penetrating peptide (CPP) conjugate, such as TAT-GRK2i, or use a viral vector to express the GRK2i peptide sequence (e.g., AAV-GRK2i). For TAT-GRK2i in mice: Adult mice (e.g., C57BL/6J, 8-12 weeks old) are given an intraperitoneal (i.p.) or intravenous (i.v.) injection of TAT-GRK2i (1-10 mg/kg in sterile saline) daily for 1-14 days. For cardiac studies, myocardial infarction (MI) or pressure overload (transverse aortic constriction, TAC) models are used. TAT-GRK2i (2-5 mg/kg, i.p.) is administered daily starting 1 day before surgery and continued for 2-4 weeks after surgery. Endpoints include echocardiography (ejection fraction, fractional shortening), heart weight-to-body weight ratio, fibrosis (Masson's trichrome staining), and Western blot for GRK2 and Gbetagamma signaling. For neurological studies, TAT-GRK2i is administered by intracerebroventricular (i.c.v.) injection (2-5 uL of 100-500 uM solution) or via osmotic minipump. Pharmacodynamic effects are assessed by behavioral tests (e.g., open field, tail suspension, forced swim test) and tissue collection for immunohistochemistry and Western blot. For ex vivo analysis: tissues (heart, brain, liver) are harvested, homogenized, and GRK2 activity or Gbetagamma signaling (e.g., p-AKT, p-ERK) is measured by Western blot. All animal procedures must be approved by IACUC. The TFA salt form is for research use only, not for clinical use. The GRK2i peptide is not approved as a drug.

No specific pharmacokinetic (PK) data are available for GRK2i TFA. As a 29-amino acid peptide (MW ~3.6 kDa), GRK2i is not orally bioavailable and is rapidly cleared from the circulation (plasma half-life of <5-15 minutes) due to renal filtration and proteolytic degradation. To improve pharmacokinetics, the peptide is often conjugated to a cell-penetrating peptide (e.g., TAT, penetratin) or encapsulated in nanoparticles. The TAT-GRK2i conjugate has an extended half-life compared to the naked peptide but still has rapid clearance (t1/2 15-30 min in mice). The TFA salt form is used during synthesis and does not alter the PK profile. For PK studies: Administer GRK2i or TAT-GRK2i (5 mg/kg, i.v.) to mice, collect blood at 0, 5, 15, 30, 60, 120 min, and quantify by LC-MS/MS. The compound distributes to tissues including heart, kidney, and liver, but brain penetration is poor (<5% of plasma). The peptide is primarily cleared by the kidneys (glomerular filtration) and metabolized by tissue peptidases. Detailed PK parameters are not publicly available. GRK2i is not a drug candidate; it is a research tool.

No specific toxicity data are available for GRK2i TFA. In vitro, GRK2i (up to 100 uM) is generally not cytotoxic to cells as measured by MTT or LDH release assays, although high concentrations (>50 uM) may cause non-specific effects. In vivo, TAT-GRK2i administered at 1-10 mg/kg i.p. daily for 2-4 weeks is generally well-tolerated, with no reported mortality or significant weight loss, though some studies report mild lethargy. No genotoxicity, organ toxicity, or carcinogenicity data exist. Since GRK2i sequesters Gbetagamma subunits, which are involved in many cellular functions, chronic inhibition might cause adverse effects; however, no such studies have been reported. The TFA salt is present in low amounts and is considered non-toxic. GRK2i is for research use only and is not approved for human use. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be used. The compound is not intended for therapeutic applications.

[1]. Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction. BMC Neurosci. 2014 Dec 31;15:132.

GRK2 (G protein-coupled receptor kinase 2) is a serine/threonine kinase that plays a critical role in homologous desensitization and internalization of GPCRs, including beta-adrenergic receptors (beta-AR). GRK2 is overexpressed in heart failure, hypertension, and chronic inflammation, making it a therapeutic target for cardiovascular diseases. GRK2 is activated by binding to free Gbetagamma dimers released upon GPCR activation. The Gbetagamma-binding domain resides in the N-terminal region of GRK2 (the PH domain). GRK2i is a 29-amino acid peptide that corresponds to this Gbetagamma-binding domain. It acts as a competitive inhibitor of Gbetagamma binding to GRK2 and other effectors. GRK2i was first described by Koch and colleagues (1994-1995). The peptide sequence is WKKELRDAYREAQQLVQRVPKMKNKPRS (positions 50-78 of bovine GRK2). The TFA salt is used to stabilize the peptide during lyophilization and storage. GRK2i has been used extensively as a research tool to dissect Gbetagamma-dependent signaling pathways in cardiac myocytes, neurons, neutrophils, and cancer cells. However, the peptide itself is not approved as a therapeutic; cell-permeable TAT-GRK2i conjugates have been tested in preclinical models of heart failure and inflammation. As of 2026, no GRK2i-based therapy has been approved for clinical use. This product is for research use only.

C152H257F3N50O43S

3598.10

GRK2i;148505-03-7

White to off-white solid powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~100 mg/mL (~27.79 mM)

Solubility in Formulation 1: 50 mg/mL (13.90 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)