Extraction and Purification of Functional G Protein-Coupled Receptors from Expression Systems - CN (2023)

Extraction and Purification of Functional G Protein-Coupled Receptors from Expression Systems

byJoanna Geddes, Chris Wojewodzki, Barbara Kaboord, and Kay Opperman

G protein-coupled receptors (GPCRs), spanning 7-transmembrane domains, are a large protein family of receptors that play a key role in transmitting signals across the cell membrane. The requirement for stabilized G protein-coupled receptors (GPCRs) in their native, functional form presents significant challenges for in vitro functional and structural characterization. Since endogenous GPCR expression levels are typically very low, most researchers elect to overexpress their GPCR of interest in order to obtain the sample concentration needed for structural analysis. There are many considerations when choosing your expression system. While E. coli and insect expression systems may yield more protein, it has been shown that mammalian systems are more ideal to mimic native conformation of receptors for functional and structural analysis [1,2]. Based on the expression system chosen, adjustments to the extraction protocol are required to get the most efficient solubilization of functional receptor. Maintaining solubility and native conformation of overexpressed GPCR constructs throughout the downstream purification process is also challenging. Therefore, optimization of the affinity purification protocol and incorporation of stabilization factors into the buffer systems are required to maintain receptor functionality throughout the sample preparation [3,4,5].

Here we report an optimized method of extraction and purification of adenosine receptor type 2A (A2AR) that yields purified receptor ready for functional analysis. Extraction of A2AR using the GPCR Extraction and Stabilization Reagent yields a stable receptor in whole cell lysate that can be stored for up to 1 week at 4⁰C or 1 month at -20⁰C. The extracted receptor can then be processed further using affinity purification appropriate for the tagged target. The addition of the GPCR Extraction and Stabilization Reagent diluted 10-fold into the recommended wash and elution buffers aids in maintaining receptor stability throughout the entire purification process.


Protein Extraction

Whole Cell Protein Extraction: Fresh or frozen pelleted Expi293 cells (1x107) expressing C-terminally His-tagged A2AR were washed in 1 mL PBS and pelleted at 500 x g for 5 minutes. After the supernatant was discarded, the cell pellet was subsequently suspended in 1 mL of cold GPCR Extraction and Stabilization Reagent (Cat No. A43436) by either gently pipetting up and down, using a dounce homogenizer (25–50 strokes), or sonicating at 30% Amps for 10 seconds to lyse. Cells were then incubated for 60 minutes at 4ºC with end-over-end mixing. After incubation, samples were centrifuged at 16,000 x g for 20 minutes at 4ºC to pellet cell debris. The supernatant containing the solubilized protein was collected. Protein concentration was determined using the Pierce Rapid Gold BCA Protein Assay Kit (Cat No. A53225).

Ni-NTA Purification

Pierce Ni-NTA Magnetic Agarose Beads (Cat No. 78605) (100 µL of a 25% slurry) were pipetted into a 1.5 mL microcentrifuge tube for each sample. The beads were then washed twice with 500 µL of cold Equilibration Buffer (25 mM HEPES, 0.3 M NaCl, 10 mM imidazole, mixed 9:1 with GPCR Extraction and Stabilization Reagent, pH 8.0). Protein extract (1 mL diluted to 1 mg/mL with Equilibration Buffer) was added to the washed beads, vortexed for 10 seconds, and then mixed on an end-over-end rotator for 60 minutes at 4ºC. After incubation, the beads were collected on a magnetic stand, and the supernatant was saved (flow-through) for downstream analysis. Next, the beads with the bound A2AR-GFP-His were washed twice with 500 µL of Wash Buffer (25 mM HEPES, 0.3 M NaCl, 15 mM imidazole, mixed 9:1 with GPCR Extraction and Stabilization Reagent, pH 8.0). Bound receptor was eluted from the beads by adding 250 µL of Elution Buffer (25 mM HEPES, 0.3 M NaCl, 0.3 M imidazole, mixed 9:1 with GPCR Extraction and Stabilization Reagent, pH 8.0) to the washed beads and then incubated on an end-over-end rotator for 10 minutes at room temperature.
Note: Thermostability may vary by receptor, depending on your target, an elution at 4ºC with increased incubation time may be necessary. Beads were then collected on a magnetic stand, and the eluate containing A2AR-GFP-His was carefully removed. Samples were either directly analyzed by SDS-PAGE and Silver Stain or buffer exchanged into the GPCR Extraction and Stabilization Reagent for overnight storage at 4ºC using Thermo Scientific Zeba Spin Desalting Columns before proceeding to radio-ligand binding assays.

Radioligand Binding Assay

A2AR-GFP-His starting lysate, flow-through, and purified samples were prepared from frozen Expi293 cell pellets. For the binding assay, 5 μg of each sample were incubated for 75 minutes at room temperature with either 3H-Adenosine alone to determine total binding, or with unlabeled adenosine and 3H-Adenosine to determine non-specific binding. Free radioligand was then removed using Pierce Dye and Biotin Removal Spin Columns, and bound radioligand was then quantified using a TRI-CARB 2000 TR scintillation counter.


Click image to enlarge

Figure 1: When comparing different homogenization methods for extraction of functional A2AR, the following trends were observed:
1) Increased agitation improved protein yield.
2) Dounce homogenized samples yielded the highest amount of active A2AR.
3) Sonication increased protein yield but decreased the amount of active receptor.

Click image to enlarge

Figure 2: The ability to purify A2AR-GFP-His in the presence of GPCR Extraction and Stabilization Reagent at 1/10 strength successfully stabilized the receptor throughout the process.

Figure 3: Our purification protocol demonstrated enriched activity of A2AR-GFP-His in the final eluate. When comparing equal amounts of starting protein load (5 µg) to the eluate samples, there is a 4-fold increase in activity following purification.

Click image to enlarge


Using agitation at the beginning of the lysis incubation will increase protein yield; however, too much, as in the case with sonication, affects the conformation of the target protein. We recommend using gentle agitation at this step using a dounce or handheld tissue homogenizer depending on sample volume (Figure 1). The addition of GPCR Extraction and Stabilization Reagent at 1/10 strength into the purification buffer system does not negatively influence the binding of A2AR-GFP-His to the Ni-NTA magnetic agarose beads (Figure 2). This addition is important to keep the critical micelle concentration constant as it plays a crucial role in maintaining functionality of A2AR-GFP-His throughout the entire purification process. This is demonstrated when comparing specific activity of equal amounts of starting lysate, flow-through, and eluates. A significant enrichment (>4-fold) of purified active receptor is observed (Figure 3). With evolving techniques and technologies, such as native mass spectrometry and cryo-EM, extraction of a stable receptor will be essential. With the improved receptor stability that the GPCR Extraction and Stabilization Reagent provides in both the extraction and purification methods, researchers will have a greater flexibility and confidence in their GPCR research and targeted therapeutic studies.


  1. Chiu, ML, Tsang, C, Grihalde, N, et al (2008) Over-Expression, Solubilization, and Purification of G Protein-Coupled Receptors for Structural Biology. Combinatorial Chemistry & High Throughput Screening 11: 439-462.
  2. Wiseman, DN, Otchere, A, Patel, JH, et al (2020) Expression and purification of recombinant G protein-coupled receptors: A review. Protein Expression and Purification 167: 105524.
  3. Dore, AS, Robertson, N, Errey, JC, et al (2011) Structure of the Adenosine A2A Receptor in Complex with ZM24138S and the Xanthines XAC and Caffeine. Structure 19: 1283-1293.
  4. Corin, K, Tegler, LT, Koutsopoulos, S (2014) G-Protein-Coupled Receptor Expression and Purification. Methods in Molecular Biology 1129: 461-489.
  5. O’Malley, MA, Helgeson, ME, Wagner, NJ, et al (2011) Toward Rational Design of Mixed Micelles That are critical for the In Vitro Stabilization of G-Protein-Coupled Receptor. Biophysical Journal 101: 1938-1948.


What are the 4 steps in G protein coupled receptor activation? ›

The most important steps are (1) agonist binding, (2) receptor conformational change, (3) receptor–G-protein interaction, (4) G-protein conformational changes including GDP release and GTP binding, (5) G protein–effector interaction, (6) change in effector activity and (7) the resulting ion conductance or second ...

What are G-protein-coupled receptors and how do they work? ›

G protein–coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into α and βγ subunits that mediate downstream signals.

What processes are accomplished by G-protein-coupled receptors? ›

Introduction. G protein coupled receptors (GPCRs) are integral membrane proteins that are used by cells to convert extracellular signals into intracellular responses, including responses to hormones, neurotransmitters, as well as responses to vision, olfaction and taste signals.

How do you activate G-protein-coupled receptors? ›

Binding of specific ligands, such as hormones, neurotransmitters, chemokines, lipids, and glycoproteins, activates GPCRs by inducing or stabilizing a new conformation in the receptor (1, 2).

What is the process of the G-protein receptor? ›

G proteins relay their signal on the inside surface of the cell membrane. The process starts when a receptor binds to its proper hormone or neurotransmitter, such as adrenaline (shown on the left using PDB entry 3sn6). This changes the shape of the receptor, and it binds to the inactive three-chain G protein inside.

What is the effect of activating G-protein coupled neurotransmitter receptors? ›

The effector protein activated by the G-protein can create many second messengers, and the activated protein kinases can each phosphorylate multiple cellular proteins. This means that one neurotransmitter can have a significant effect on cellular function.

Which neurotransmitters use G protein coupled receptors? ›

Common GPCR ligands in the nervous system are: Monoamines: adrenaline, noradrenaline, serotonin, dopamine, histamine. Other small neurotransmitters: Acetylcholine (mACh), gamma aminobutyric acid (GABAB), glutamate (metabotropic, mGluR), ATP (P2Y), adenosine, cannabinoids.

What is the role of G proteins in G protein coupled receptors? ›

Heterotrimeric G-proteins mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues and organs, these signals modulate diverse physiological functions.

How are G protein coupled receptors regulated? ›

GPCR signaling is tightly regulated by various mechanisms, including internalization, desensitization, and interaction with cytosolic proteins. These mechanisms are influenced by posttranslational modifications such as phosphorylation, glycosylation, palmitoylation and ubiquitination [15,16,17,18].

What enzyme is activated by G protein coupled receptors? ›

Many G-protein-linked receptors exert their effects mainly via G proteins that activate the plasma-membrane-bound enzyme phospholipase C-β.

How are G proteins activated and regulated? ›

Heterotrimeric G proteins located within the cell are activated by G protein-coupled receptors (GPCRs) that span the cell membrane. Signaling molecules bind to a domain of the GPCR located outside the cell, and an intracellular GPCR domain then in turn activates a particular G protein.

How do G proteins become activated? ›

G proteins are molecular switches that are activated by receptor-catalyzed GTP for GDP exchange on the G protein alpha subunit, which is the rate-limiting step in the activation of all downstream signaling.

What drugs binds to G protein coupled receptors? ›

Hence, there is enormous potential for the development of new drugs targeting these receptors. Examples of drugs targeting GPCRs include histamine receptor blockers, opioid agonists, β-blockers and angiotensin receptor blockers.

What are the 7 g protein coupled receptors? ›

G protein-coupled receptors, or GPCRs, also known as 7-Transmembrane receptors (7-TM receptors), are integral membrane proteins that contain seven membrane-spanning helices. As the name suggests they are coupled to heterotrimeric G proteins on the intracellular side of the membrane.

Why are G protein coupled receptors good drug targets? ›

GPCRs have been a major target for drug developers because of their regulation of a wide variety of human physiological processes, including growth, metabolism and homeostasis.

Where are G protein coupled receptors synthesized? ›

The life of GPCRs begins at the ER where they are synthesized, folded and assembled. Properly folded receptors are recruited and packaged into ER-derived COPII-coated vesicles.

Where are G protein coupled receptors? ›

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that are located in the cell membrane, with their N- and C-termini located on the outer and inner surfaces, respectively. GPCRs mediate various cellular responses from the extracellular environment.

What are the different types of G protein coupled receptors? ›

GPCRs are categorized into six classes based on sequence and function, namely Class A—rhodopsin-like receptors, Class B—secretin family, Class C—metabotropic glutamate receptors, Class D—fungal mating pheromone receptors, Class E—cAMP receptors, and Class F—frizzled (FZD) and smoothened (SMO) receptors (Lee et al., ...

What type of hormones activates G proteins? ›

The amino acid-derived hormones epinephrine and norepinephrine bind to beta-adrenergic receptors on the plasma membrane of cells. Hormone binding to receptor activates a G-protein, which in turn activates adenylyl cyclase, converting ATP to cAMP. cAMP is a second messenger that mediates a cell-specific response.

How are G proteins activated and inactivated? ›

The activation occurs by conversion of G-protein alpha (Gα)-coupled guanosine diphosphate (GDP) to Guanosine-5-triphosphate (GTP). The activated G-protein then dissociates into an α and a β/γ complex. GTP bound Gα is active. Intrinsic GTPase activity leads to the inactivation of the G-Protein.

How are G proteins converted from the inactive to the active state? ›

In the inactive state, the G protein binds the nucleotide GDP. The G-protein has three subunits, alpha, beta and gamma. Activation of the receptor by the neurotransmitter dopamine causes the alpha subunit to exchange its GDP for a GTP.

What is the primary role of G protein in the cell? ›

Heterotrimeric G proteins are major signaling mediators, which play an important role in propagating the extracellular information to cell interior. Agonist-bound GPCR undergoes conformational changes and subsequent heterotrimer dissociation.

What is the G protein coupled receptor cycle? ›

The G protein-coupled receptor is activated by an external signal in the form of a ligand or other signal mediator. This creates a conformational change in the receptor, causing activation of a G protein. Further effect depends on the type of G protein.

What is the first step of GPCR activation? ›

In inactivation state the GDP is bound to Gα (Gα-GDP). In signal transduction, first the GPCR gets activated by changing its conformation which resulted from binding of agonist/ligands to the extracellular region of GPCR.

How does a G-protein become activated? ›

G proteins are molecular switches that are activated by receptor-catalyzed GTP for GDP exchange on the G protein alpha subunit, which is the rate-limiting step in the activation of all downstream signaling.

What are the general steps of GPCR signaling quizlet? ›

  • First messenger binds to and activates a membrane receptor.
  • Gs protein associates with activated receptor.
  • GDP dissociates from Gs protein.
  • GTP binds to Gs protein.
  • Gs protein dissociates into α and β /γ subunits.
  • α subunit of Gs protein binds to and activates adenylate cyclase.

What hormones bind G protein coupled receptors? ›

Class B G-protein-coupled receptors (GPCRs) are receptors for peptide hormones that include glucagon, parathyroid hormone, and calcitonin. These receptors are involved in a wide spectrum of physiological activities, from metabolic regulation and stress control to development and maintenance of the skeletal system.

What enzyme does G-protein activate? ›

One especially common target of activated G proteins is adenylyl cyclase, a membrane-associated enzyme that, when activated by the GTP-bound alpha subunit, catalyzes synthesis of the second messenger cAMP from molecules of ATP.

What is the role of G-protein in cell signaling? ›

The main physiological functions of G-proteins are to relay the signals from GPCRs which function as GEFs for G-proteins. Binding with exogenous or endogenous agonists induces GPCRs into an active conformational state which, in turn, influences intracellular binding of G-proteins or arrestin proteins [23, 24].

What is the 3 types of G-protein? ›

G protein coupled receptors (GPCRs) are one of the major classes of cell surface receptors and are associated with a group of G proteins consisting of three subunits termed alpha, beta, and gamma.

What is the main event that will activate the G proteins? ›

They are activated in response to a conformational change in the GPCR, exchanging GDP for GTP, and dissociating in order to activate other proteins in a particular signal transduction pathway. The specific mechanisms, however, differ between protein types.

Where are G-protein-coupled receptors located? ›

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that are located in the cell membrane, with their N- and C-termini located on the outer and inner surfaces, respectively. GPCRs mediate various cellular responses from the extracellular environment.

What types of G proteins regulate GPCR Signalling? ›

Signaling by G protein-coupled receptors (GPCRs) and arrestins. Agonist-activated GPCRs (agonist is shown as a green ball) bind heterotrimeric G proteins, serving as GEFs: they facilitate the release of GDP bound to the α-subunit of inactive heterotrimer, which subsequently bind GTP.

What are two major signal transduction mechanisms triggered by G protein coupled receptors? ›

The general mechanism involved in cellular communication has been described. However, two, main signal transduction pathways involve GPCRs that can be discussed in more detail. These two pathways are the cAMP signal pathway and the phosphatidylinositol signal pathway.


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