G protein-coupled receptor (GPCR) signaling is regulated by four ubiquitously expressed GPCR kinase isoforms (GRKs), namely GRK2, GRK3, GRK5, and GRK6. Overexpression of individual GRKs occurs in diseases like cancer and heart failure, prompting a search for potent GRK inhibitors. While various in silico and in vitro approaches exist, few methods assess inhibitor efficacy in cellular systems. To address this, we used HEK293 cell lines co-expressing the β2 adrenergic receptor (β2) and one GRK isoform on a quadruple GRK2/3/5/6 knockout background (ΔQ-GRK). We evaluated the inhibition of isoproterenol (ISO)-induced T360/S364-β2 phosphorylation using the 7TM phosphorylation assay. This combination allowed comprehensive evaluation of commercially available GRK inhibitors. We conclude that compound 8h (GRK2/3 inhibitor) and compound 18 (GRK5/6 inhibitor) are highly recommendable tools for the study of GPCR phosphorylation and function in cellular systems. Together, these cell-based GRK inhibitor assays can facilitate medium- to high-throughput screening of future GRK-targeted drug candidates. A GRK-isoform immunoassay was used to quantify GPCR phosphorylation and evaluate the potency of GRK inhibitors in a controlled cellular system.

Desensitization and alternative signaling pathways of G protein-coupled receptors (GPCRs) are largely mediated by β-arrestins (arr). While most GPCRs recruit arrestin through phosphorylated C-terminal tails, many lack a canonical tail and instead rely on phosphorylation sites within long third intracellular loops (ICL3). Structural information on such complexes is largely missing, and the molecular details of arrestin engagement remain unknown. Here, we dissect the interaction between the muscarinic acetylcholine receptor M2 (M2R) and β-arrestins directly in living cells using genetically encoded crosslinkers and derive crosslinking-guided atomistic models of the M2R-arrestin complex, supported by unbiased molecular dynamics simulations. Our results show that the M2R ICL3 wraps around a positively charged belt on the arrestin N-domain, whereas the receptor core engages the arrestin central crest through a highly dynamic interface. This core contact is essential for efficient arrestin binding and is tuned by the topology of phosphorylation sites, with markedly stronger engagement when the sites reside in a C-terminal tail rather than in ICL3. Our work provides previously inaccessible structural insight into GPCR-arrestin complexes that rely on ICL3-mediated recruitment and suggests that the topology of the interaction, rather than sequence motifs alone, governs the functional dynamics of arrestin coupling.

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

Agonist-induced phosphorylation is a crucial step in the activation/deactivation cycle of G protein-coupled receptors (GPCRs), but direct determination of individual phosphorylation events has remained a major challenge. We have recently developed a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in 96-well plates, thus eliminating the need for western blot analysis. In the present study, we adapted this assay to three novel phosphosite-specific antibodies directed against the neurokinin 1 (NK1) receptor, namely pS338/pT339-NK1, pT344/pS347-NK1, and pT356/pT357-NK1. We found that substance P (SP) stimulated concentration-dependent phosphorylation of all three sites, which could be completely blocked in the presence of the NK1 receptor antagonist aprepitant. The other two endogenous ligands of the tachykinin family, neurokinin A (NKA) and neurokinin B (NKB), were also able to induce NK1 receptor phosphorylation, but to a much lesser extent than substance P. Interestingly, substance P promoted phosphorylation of the two distal sites more efficiently than that of the proximal site. The proximal site was identified as a substrate for phosphorylation by protein kinase C. Analysis of GPCR kinase (GRK)-knockout cells revealed that phosphorylation was mediated by all four GRK isoforms to similar extents at the T344/S347 and the T356/T357 cluster. Knockout of all GRKs resulted in abolition of all phosphorylation signals highlighting the importance of these kinases in agonist-mediated receptor phosphorylation. Thus, the 7TM phosphorylation assay technology allows for rapid and detailed analyses of GPCR phosphorylation.

Free fatty acid receptor 2 (FFAR2) is activated by short-chain fatty acids and expressed widely, including in white adipocytes and various immune and enteroendocrine cells. Using both wild-type human FFAR2 and a designer receptor exclusively activated by designer drug (DREADD) variant we explored the activation and phosphorylation profile of the receptor, both in heterologous cell lines and in tissues from transgenic knock-in mouse lines expressing either human FFAR2 or the FFAR2-DREADD. FFAR2 phospho-site-specific antisera targeting either pSer296/pSer297 or pThr306/pThr310 provided sensitive biomarkers of both constitutive and agonist-mediated phosphorylation as well as an effective means to visualise agonist-activated receptors in situ. In white adipose tissue, phosphorylation of residues Ser296/Ser297 was enhanced upon agonist activation whilst Thr306/Thr310 did not become phosphorylated. By contrast, in immune cells from Peyer’s patches Thr306/Thr310 become phosphorylated in a strictly agonist-dependent fashion whilst in enteroendocrine cells of the colon both Ser296/Ser297 and Thr306/Thr310 were poorly phosphorylated. The concept of phosphorylation bar-coding has centred to date on the potential for different agonists to promote distinct receptor phosphorylation patterns. Here, we demonstrate that this occurs for the same agonist-receptor pairing in different patho-physiologically relevant target tissues. This may underpin why a single G protein-coupled receptor can generate different functional outcomes in a tissue-specific manner.

G protein-coupled receptors (GPCRs) are important signal transducers that are phosphorylated upon activation at intracellular serine and threonine residues. Although antibodies that specifically recognize the phosphorylation state of GPCRs have been available for many years, efficient immunolocalization of phosphorylated receptors in their tissues of origin has not been possible. Here, we show that phosphorylation of receptors is highly unstable during routine immunohistochemical procedures, requiring the use of appropriate phosphatase inhibitors particular during tissue perfusion, post-fixation, and cryoprotection but not during immunostaining of tissue sections. We provide proof of concept using phosphorylation state-specific μ-opioid receptor (MOP) and cannabinoid receptor 1 (CB1) antibodies. Indeed, three of four well-characterized phosphosite-specific MOP antibodies, including pS375-MOP, pT376-MOP, and pT379-MOP, showed robust neuronal immunostaining in brain and spinal cord sections of opioid-treated mice only after inclusion of phosphatase inhibitors. We then extended this approach to the CB1 receptor and demonstrated that one of three newly-generated phosphosite-specific CB1 antibodies, namely pS425-CB1, showed striking staining of fibers and varicosities in brain slices from cannabinoid-treated mice. Although subsequent experiments showed that phospho-CB1 immunostaining was less sensitive to phosphatases, we conclude that the use of phosphatase inhibitors should always be considered in the development of immunohistochemical procedures for new phosphosite-specific GPCR antibodies. In summary, we anticipate that this improved protocol will facilitate the widespread use of phosphorylation state-specific antibodies to monitor the activation of endogenous GPCRs under physiological and pharmacological conditions. Our approach may also prove useful to confirm target engagement of GPCR drug candidates in native tissues.

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.

GPR84 is an immune cell-expressed, proinflammatory receptor currently being assessed as a therapeutic target in conditions including fibrosis and inflammatory bowel disease. Although it was previously shown that the orthosteric GPR84 activators 2-HTP and 6-OAU promoted its interactions with arrestin-3, a G protein-biased agonist DL 175 did not. Here, we show that replacement of all 21 serine and threonine residues within i-loop 3 of GPR84, but not the two serines in the C-terminal tail, eliminated the incorporation of [P-32] and greatly reduced receptor- arrestin-3 interactions promoted by 2-HTP. GPR84 was phosphorylated constitutively on residues Ser(221) and Ser(224), while various other amino acids are phosphorylated in response to 2-HTP. Consistent with this, an antiserum able to identify pSer(221)/pSer(224 )recognized GPR84 from cells treated with and without activators, whereas an antiserum able to identify pThr(263)/pThr(264) only recognized GPR84 after exposure to 2-HTP and not DL-175. Two distinct GPR84 antagonists as well as inhibition of G protein- coupled receptor kinase 2/3 prevented phosphorylation of pThr(263)/pThr(264), but neither strategy affected constitutive phosphorylation of Ser(221)/Ser(224). Furthermore, mutation of residues Thr263 and Thr264 to alanine generated a variant of GPR84 also limited in 2-HTP-induced interactions with arrestin-2 and-3. By contrast, this mutant was unaffected in its capacity to reduce cAMP levels. Taken together, these results define a key pair of threonine residues, regulated only by subsets of GPR84 small molecule activators and by GRK2/3 that define effective interactions with arrestins and provide novel tools to monitor the phosphorylation and functional status of GPR84.

The human somatostatin receptor 3 (sst3) is expressed in about 50% of all neuroendocrine tumors and hence a promising target for multireceptor somatostatin analogs. The sst3 receptor is unique among ssts in that it exhibits a very long intracellular C-terminal tail containing a huge number of potential phosphate acceptor sites. Consequently, our knowledge about the functional role of the C-terminal tail in sst3 receptor regulation is very limited. Here, we have generated a series of phosphorylation-deficient mutants that enabled us to determine crucial sites for its agonist-induced β-arrestin mobilization, internalization, and down-regulation. Based on this information, we generated phosphosite-specific antibodies for C-terminal Ser(337)/Thr(341), Thr(348), and Ser(361) that enabled us to investigate the temporal patterns of sst3 phosphorylation and dephosphorylation. We found that the endogenous ligand somatostatin induced a rapid and robust phosphorylation that was completely blocked by the sst3 antagonist NVP-ACQ090. The stable somatostatin analogs pasireotide and octreotide promoted clearly less phosphorylation compared with somatostatin. We also show that sst3 phosphorylation occurred within seconds to minutes, whereas dephosphorylation of the sst3 receptor occurred at a considerable slower rate. In addition, we also identified G protein-coupled receptor kinases 2 and 3 and protein phosphatase 1α and 1β as key regulators of sst3 phosphorylation and dephosphorylation, respectively. Thus, we here define the C-terminal phosphorylation motif of the human sst3 receptor that regulates its agonist-promoted phosphorylation, β-arrestin recruitment, and internalization of this clinically relevant receptor.

The biological actions of somatostatin are mediated by a family of five GPCRs , named sst 1 to sst 5 . Somatostatin receptors exhibit equally high‐binding affinities to their natural ligand somatostatin‐14 and largely overlapping distributions. The overexpression of somatostatin receptors in human tumours is the molecular basis for diagnostic and therapeutic application of the stable somatostatin analogues octreotide, lanreotide and pasireotide. The efficiency of somatostatin receptor signalling is tightly regulated and ultimately limited by the coordinated phosphorylation and dephosphorylation of intracellular carboxyl‐terminal serine and threonine residues. Here, we review and discuss recent progress in the generation and application of phosphosite‐specific antibodies for human sst 2 and sst 5 receptors. These phosphosite‐specific antibodies are unique tools to monitor the spatial and temporal dynamics of receptors phosphorylation and dephosphorylation. Using a combined approach of phosphosite‐specific antibodies and si RNA knock‐down screening, relevant kinases and phosphatases were identified. Emerging evidence suggests distinct mechanisms of agonist‐selective fine‐tuning for individual somatostatin receptors. The recently uncovered differences in phosphorylation and dephosphorylation of these receptors may hence be of physiological significance in mediating responses to acute, persistent or repeated stimuli in a variety of target tissues.

The somatostatin receptor 2 (sst2) is the pharmacological target of somatostatin analogs that are widely used in the diagnosis and treatment of human neuroendocrine tumors. We have recently shown that the stable somatostatin analogs octreotide and pasireotide (SOM230) stimulate distinct patterns of sst2 receptor phosphorylation and internalization. Like somatostatin, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359, which in turn leads to a robust receptor endocytosis. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor followed by a partial receptor internalization. Here, we show that exchange of S341 and S343 by alanine is sufficient to block pasireotide-driven internalization, whereas mutation of T353, T354, T356 and T359 to alanine is required to strongly inhibited both octreotide- and somatostatin-induced internalization. Yet, combined mutation of T353, T354, T356 and T359 is not sufficient to prevent somatostatin-driven β-arrestin mobilization and receptor desensitization. Replacement of all fourteen carboxyl-terminal serine and threonine residues by alanine completely abrogates sst2 receptor internalization and β-arrestin mobilization in HEK293 cells. Together, our findings demonstrate for the first time that agonist-selective sst2 receptor internalization is regulated by multi-site phosphorylation of its carboxyl-terminal tail.

We have recently identified protein phosphatase 1β (PP1β) as G protein-coupled receptor (GPCR) phosphatase for the sst2 somatostatin receptor using siRNA knockdown screening. By contrast, for the sst5 somatostatin receptor we identified protein phosphatase 1γ (PP1γ) as GPCR phosphatase using the same approach. We have also shown that sst2 and sst5 receptors differ substantially in the temporal dynamics of their dephosphorylation and trafficking patterns. Whereas dephosphorylation and recycling of the sst2 receptor requires extended time periods of ∼30 min, dephosphorylation and recycling of the sst5 receptor is completed in less than 10 min. Here, we examined which receptor domains determine the selection of phosphatases for receptor dephosphorylation. We found that generation of tail-swap mutants between sst2 and sst5 was required and sufficient to reverse the patterns of dephosphorylation and trafficking of these two receptors. In fact, siRNA knockdown confirmed that the sst5 receptor carrying the sst2 tail is predominantly dephosphorylated by PP1β, whereas the sst2 receptor carrying the sst5 tail is predominantly dephosphorylated by PP1γ. Thus, the GPCR phosphatase responsible for dephosphorylation of individual somatostatin receptor subtypes is primarily determined by their different carboxyl-terminal receptor domains. This phosphatase specificity has in turn profound consequences for the dephosphorylation dynamics and trafficking patterns of GPCRs.

Background: The somatostatin receptor 1 (sst(1)) is widely distributed throughout the body and is also present in neoplastic tissues. However, little is known about its precise tissue distribution, regulation and function, which may in part be due to the lack of specific monoclonal anti-sst(1) antibodies.Methods: We have characterized the novel rabbit monoclonal anti-human sst(1) antibody UMB-7 using sst(1)-expressing cells and human pituitary samples. The antibody was then used for immunohistochemical staining of a large panel of formalin-fixed, paraffin-embedded human tissues.Results: Western blot analyses of BON-1 cells and human pituitary revealed a broad band migrating at a molecular weight of 45,000-60,000. After enzymatic deglycosylation the size of this band decreased to a molecular weight of 45,000. UMB-7 yielded an efficient immunostaining of distinct cell populations in the human tissue samples with a predominance of plasma membrane staining, which was completely abolished by preadsorption of UMB-7 with its immunizing peptide. The sst(1) receptor was detected in anterior pituitary, pancreatic islets, distal tubules, enteric ganglion cells and nerve fibers, chief cells of the gastric mucosa, macrophages and mast cells. In addition, sst(1) was observed in pituitary adenomas, gastrointestinal neuroendocrine tumors and pheochromocytoma as well as in pancreatic adenocarcinomas, gastric carcinomas, urinary bladder carcinomas and sarcomas.Conclusions: UMB-7 may prove of great value in the identification of sst(1)-expressing tumors during routine histopathological examinations. This may open up new routes for diagnostic and therapeutic intervention. (C) 2013 Elsevier B.V. All rights reserved.

The chemokine receptor CXCR4 regulates cell migration during ontogenesis and disease states including cancer and inflammation. Upon stimulation by the endogenous ligand CXCL12, CXCR4 becomes phosphorylated at multiple sites in its C-terminal domain. Mutations in the CXCR4 gene affecting C-terminal phosphorylation sites are a hallmark of WHIM syndrome, a genetic disorder characterized by a gain-of-CXCR4-function. To better understand how multi-site phosphorylation of CXCR4 is organized and how perturbed phosphorylation might affect CXCR4 function, we developed novel phosphosite-specific CXCR4 antibodies and studied the differential regulation and interaction of three C-terminal phosphorylation sites in human embryonic kidney cells (HEK293). CXCL12 promoted a robust phosphorylation at S346/347 which preceded phosphorylation at S324/325 and S338/339. After CXCL12 washout, the phosphosites S338/339 and S324/325 were rapidly dephosphorylated whereas phosphorylation at S346/347 was long-lasting. CXCL12-induced phosphorylation at S346/347 was staurosporine-insensitive and mediated by GRK2/3. WHIM syndrome-associated CXCR4 truncation mutants lacking the S346/347 phosphosite and the recently identified E343K WHIM mutant displayed strongly impaired phosphorylation at S324/325 and S338/339 as well as reduced CXCL12-induced receptor internalization. Relevance of the S346-S348 site was confirmed by a S346-348A mutant showing strongly impaired CXCL12-promoted phosphorylation at S324/325 and S338/339, defective internalization, gain of calcium mobilization, and reduced desensitization. Thus, the triple serine motif S346-S348 contains a major initial CXCR4 phosphorylation site and is required for efficient subsequent multi-site phosphorylation and receptor regulation. Hierarchical organization of CXCR4 phosphorylation explains why small deletions at the extreme CXCR4 C terminus typically associated with WHIM syndrome severely alter CXCR4 function.

The frequent overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of novel multireceptor somatostatin analogs. Although the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (T333) and 347 (T347), which enabled us to selectively detect either the T333-phosphorylated or the T347-phosphorylated form of sst5. We show that agonist-mediated phosphorylation occurs at T333, whereas T347 is constitutively phosphorylated in the absence of agonist. We further demonstrate that the multireceptor somatostatin analog pasireotide and the sst5-selective ligand L-817,818 but not octreotide or KE108 were able to promote a detectable T333 phosphorylation. Interestingly, BIM-23268 was the only sst5 agonist that was able to stimulate T333 phosphorylation to the same extent as natural somatostatin. Agonist-induced T333 phosphorylation was dose-dependent and selectively mediated by G protein-coupled receptor kinase 2. Similar to that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. However, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were rapidly completed within minutes. We also identify protein phosphatase 1γ as G protein-coupled receptor phosphatase for the sst5 receptor. Together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal T333. In addition, we identify G protein-coupled receptor kinase 2-mediated phosphorylation and protein phosphatase 1γ-mediated dephosphorylation of T333 as key regulators of rapid internalization and recycling of the human sst5 receptor.

Background: Among the five somatostatin receptors (sst1-sst5), the sst3 receptor displays a distinct pharmacological profile. Like sst2, the sst3 receptor efficiently internalizes radiolabeled somatostatin analogs. Unlike sst2, however, internalized sst3 receptors are rapidly transferred to lysosomes for degradation. Apart from this, very little is known about the clinical relevance of the sst3 receptor, which may in part be due to the lack of specific monoclonal sst3 antibodies. Methods: Here, we have extensively characterized the novel rabbit monoclonal anti-human sst3 antibody UMB-5 using transfected cells and receptor-expressing tissues. UMB-5 was then subjected to immunohistochemical staining of a series of 190 formalin-fixed, paraffin-embedded normal and neoplastic human tissues. Results: Specificity of UMB-5 was demonstrated by detection of a broad band migrating at a molecular weight of 70,000–85,000 in immunoblots from human pituitary. After enzymatic deglycosylation, the size of this band decreased to a molecular weight of 45,000. Tissue immunostaining was completely abolished by pre-adsorption of UMB-5 with its immunizing peptide. In addition, UMB-5 detected distinct cell populations in human tissues like pancreatic islands, anterior pituitary, adrenal cortex, adrenal medulla, and enteric ganglia, similar to that seen with a rabbit polyclonal antibody generated against a different carboxyl-terminal epitope of the sst3 receptor. In a comparative immunohistochemical study, UMB-5 yielded predominant plasma membrane staining in the majority of pituitary adenomas, pheochromocytomas, and a subset of neuroendocrine tumors. The sst3 receptor was also present in many glioblastomas, pancreatic, breast, cervix, and ovarian carcinomas. Conclusion: The rabbit monoclonal antibody UMB-5 may prove of great value in the identification of sst3-expressing tumors during routine histopathological examinations. Given its unique trafficking properties, these tumors may be potential candidates for sst3-directed receptor radiotherapy.