GPCR and Phospho-GPCR Antibodies

Phospho-GPCRs

Phosphorylation of GPCRs occurs on intracellular serine and threonine residues on the C-terminal tail that are unmasked due to conformational changes following agonist exposure.³ These structural changes are recognized by highly specific GPCR kinases (GRKs), as well as more general kinases such as protein kinase A (PKA). Ligand-mediated phosphorylation regulates acute receptor desensitization, arrestin recruitment, internalization, post-activation signaling and, in some cases, long-term tolerance to the agonist (Figure 1).⁴

Figure 1: Agonist-mediated GPCR internalization. Ligand binding to the GPCR results in conformational changes that are recognized by GPCR receptor kinases (GRKs). GRKs result in phosphorylation of the C-terminal tail, increasing affinity for arrestins. Arrestin binding leads to the dissociation of the heterotrimeric G protein from the GPCR and targeting to clathrin-coated pits for endocytosis with the help of the AP2 protein. Once internalized, the ligand dissociates and is typically degraded, and the GPCR is dephosphorylated by a protein phosphatase such as PP2A. The receptor can then be recycled to the plasma membrane or trafficked to lysosomes for degradation. Image made in BioRender.

Most GPCRs have multiple sites for phosphorylation, which are often not conserved, and the unique combination of phosphorylated sites, or the phosphorylation ‘barcode’, is important in determining the downstream effects and responses to stimuli.3 For example, the C-terminal tail of the mu opioid receptor has at least 4 distinct phosphorylation sites (Thr370, Ser375, Thr376, Thr379), which show differential responses to opioid stimulation.⁵ Antibodies against phosphorylated GPCRs must therefore be site-specific and highly validated to avoid cross-reactivity.

Our phosphosite-specific GPCR antibodies are designed to specifically detect agonist-activated GPCRs, using the relevant phospho-peptide as the immunogen.

Western Blot of GPCRs

Western blot is a useful technique for measuring changes in GPCR expression or the level of phosphorylation in response to pharmacological treatments. The following tips will increase the chance of developing a strong, reliable signal.

1. GPCRs should be enriched first
   Even the highest affinity antibodies may not be able to detect GPCRs from a non-enriched tissue lysate due to their naturally low abundance, and phospho-GPCRs will be a subset of the total GPCR content. Wheatgerm agglutinin (WGA) beads enrich all n-glycosylated proteins in cells, including the vast majority of GPCRs, meaning treating a lysate with WGA beads before blotting is an easy method for enrichment. An alternative is to use an epitope-tagged receptor and perform an immunoprecipitation.
2. Do not boil samples
   Boiling is a standard step in sample preparation prior to running an SDS-PAGE, but this denaturation step makes the GPCR more likely to aggregate, meaning they do not run on the gel. Instead, do not heat the samples above 60°C.
3. GPCR glycosylation results in large bands
   Broad bands or slight smears are common on western blots of GPCRs, because GPCRs are typically heavily glycosylated, which results in a spread of molecular weights rather than a sharp, tight band. This is normal.
4. Normalize phospho-GPCRs against the total amount of GPCR
   Western blots for phospho-GPCRs should be run alongside blots for the non-phospho-GPCR to normalize levels to and confirm that a treatment is only affecting phosphorylation state, not total abundance.

Figure 2: Western blot of wild type and mu opioid receptor (MOR) knockout mouse brain tissue lysate stained with Anti-Mu Opioid Receptor (phospho Ser375) Antibody (A334520). Treatment with etonitazene triggers MOR phosphorylation.

Figure 3: Western blot of HEK293 cell lysate stained with Anti-SSTR3 (phospho Ser337 + Thr341) Antibody (A334544), demonstrating dose-dependent phosphorylation of SSTR3 by SRIF.

IHC of Phospho-GPCRs

While IHC or ICC/IF for GPCRs is commonly done (and typically doesn’t suffer from the issues described above for western blots), performing these techniques on phospho-GPCRs can be much more challenging due to the transience of the phosphorylation. Endogenous phosphatases result in dephosphorylation being lost during sample preparation. The simple solution to this is to add a phosphatase inhibitor during the protocol to maintain phosphorylation state (Figure 4).⁵

See our Immunohistochemistry Protocols and ICC/IF Protocol for step-by-step instructions for performing these techniques.

Figure 4: Effect of phosphatase inhibitors on phospho-GPCR IHC results. IHC of non-phosphorylated (np) or Thr379 phosphorylated (pT379) mu opioid receptor (MOP) following treatment with methadone in the presence or absence of a protein phosphatase inhibitor (PPI). Signal is only observed in the presence of PPI. Reproduced under CC BY 4.0 from ⁵

Figure 5: ICC/IF of HEK293 cells stably expressing delta opioid receptor (DOR) stained with Anti-delta Opioid Receptor (phospho Ser363) Antibody (A334519). Treatment with DPDPE triggers DOR phosphorylation and internalization.

Figure 6: ICC/IF of HEK293 cells stably expressing Somatostatin Receptor 2 (SSTR2) stained with Anti-SSTR2 (phospho Thr356 + Thr359) Antibody (A334541). Treatment with SRIF triggers phosphorylation of SSTR2.

Applications of Phospho-GPCR Antibodies

Antibodies are an essential tool for characterizing GPCR function and responses to pharmacological manipulation, which are important to understand for the development of new therapeutics, given that GPCRs represent approximately one third of FDA-approved drugs.¹

Phosphosite-specific antibodies are novel tools for GPCR research that can be used to:

• Characterize novel GPCR ligands
• Dissect the functions of specific phosphorylated residues in signal transduction
• Determine the spatial and temporal dynamics of GPCR phosphorylation
• Identify kinases and phosphatases involved in GPCR phosphorylation and dephosphorylation

Phospho-GPCR Antibody Products

References

1. Peterson, S. M. et al. Discovery and design of G protein-coupled receptor targeting antibodies. Expert Opin. Drug Discov. 18, 417–428 (2023).
2. Chen, H., Zhang, S., Zhang, X. & Liu, H. QR code model: a new possibility for GPCR phosphorylation recognition. Cell Commun. Signal. 20, 23 (2022).
3. Tobin, A. B. G-protein-coupled receptor phosphorylation: where, when and by whom. Br. J. Pharmacol. 153, S167–S176 (2008).
4. Kliewer, A., Reinscheid, R. K. & Schulz, S. Emerging Paradigms of G Protein-Coupled Receptor Dephosphorylation. Trends Pharmacol. Sci. 38, 621–636 (2017).
5. Fritzwanker, S., Nagel, F., Kliewer, A., Stammer, V. & Schulz, S. In situ visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies. Commun. Biol. 6, 419 (2023).