Peishen Zhao

Biased signaling of protease-activated receptors.

In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain, and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an emerging therapeutic target for major diseases. Most information about PAR activation and function derives from studies of a few proteases, for example thrombin in the case of PAR1, PAR3, and PAR4, and trypsin in the case of PAR2 and PAR4. These proteases cleave PARs at established sites with the extracellular N-terminal domains, and expose tethered ligands that stabilize conformations of the cleaved receptors that activate the canonical pathways of G protein- and/or β-arrestin-dependent signaling. However, a growing number of proteases have been identified that cleave PARs at divergent sites to activate distinct patterns of receptor signaling and trafficking. The capacity of these proteases to trigger distinct signaling pathways is referred to as biased signaling, and can lead to unique patho-physiological outcomes. Given that a different repertoire of proteases are activated in various patho-physiological conditions that may activate PARs by different mechanisms, signaling bias may account for the divergent actions of proteases and PARs. Moreover, therapies that target disease-relevant biased signaling pathways may be more effective and selective approaches for the treatment of protease- and PAR-driven diseases. Thus, rather than mediating the actions of a few proteases, PARs may integrate the biological actions of a wide spectrum of proteases in different patho-physiological conditions.

The Bile Acid Receptor TGR5 Activates the TRPA1 Channel to Induce Itch in Mice

BACKGROUND & AIMS Patients with cholestatic disease have increased systemic concentrations of bile acids (BAs) and profound pruritus. The G-protein-coupled BA receptor 1 TGR5 (encoded by GPBAR1) is expressed by primary sensory neurons; its activation induces neuronal hyperexcitability and scratching by unknown mechanisms. We investigated whether the transient receptor potential ankyrin 1 (TRPA1) is involved in BA-evoked, TGR5-dependent pruritus in mice. METHODS Co-expression of TGR5 and TRPA1 in cutaneous afferent neurons isolated from mice was analyzed by immunofluorescence, in situ hybridization, and single-cell polymerase chain reaction. TGR5-induced activation of TRPA1 was studied in in HEK293 cells, Xenopus laevis oocytes, and primary sensory neurons by measuring Ca(2+) signals. The contribution of TRPA1 to TGR5-induced release of pruritogenic neuropeptides, activation of spinal neurons, and scratching behavior were studied using TRPA1 antagonists or Trpa1(-/-) mice. RESULTS TGR5 and TRPA1 protein and messenger RNA were expressed by cutaneous afferent neurons. In HEK cells, oocytes, and neurons co-expressing TGR5 and TRPA1, BAs caused TGR5-dependent activation and sensitization of TRPA1 by mechanisms that required Gβγ, protein kinase C, and Ca(2+). Antagonists or deletion of TRPA1 prevented BA-stimulated release of the pruritogenic neuropeptides gastrin-releasing peptide and atrial natriuretic peptide B in the spinal cord. Disruption of Trpa1 in mice blocked BA-induced expression of Fos in spinal neurons and prevented BA-stimulated scratching. Spontaneous scratching was exacerbated in transgenic mice that overexpressed TRG5. Administration of a TRPA1 antagonist or the BA sequestrant colestipol, which lowered circulating levels of BAs, prevented exacerbated spontaneous scratching in TGR5 overexpressing mice. CONCLUSIONS BAs induce pruritus in mice by co-activation of TGR5 and TRPA1. Antagonists of TGR5 and TRPA1, or inhibitors of the signaling mechanism by which TGR5 activates TRPA1, might be developed for treatment of cholestatic pruritus.

Cathepsin S Causes Inflammatory Pain via Biased Agonism of PAR2 and TRPV4

Background: Proteases trigger inflammation and pain by cleaving protease-activated receptors (PARs) at defined sites. Results: Cathepsin S (Cat-S) cleaved PAR2 at a unique site E56↓T57, leading to Gαs-mediated cAMP accumulation and TRPV4-dependent inflammation and pain. Conclusion: Cat-S is a biased agonist of PAR2- and TRPV4-dependent inflammation and pain. Significance: PARs integrate responses to diverse proteases. Serine proteases such as trypsin and mast cell tryptase cleave protease-activated receptor-2 (PAR2) at R36↓S37 and reveal a tethered ligand that excites nociceptors, causing neurogenic inflammation and pain. Whether proteases that cleave PAR2 at distinct sites are biased agonists that also induce inflammation and pain is unexplored. Cathepsin S (Cat-S) is a lysosomal cysteine protease of antigen-presenting cells that is secreted during inflammation and which retains activity at extracellular pH. We observed that Cat-S cleaved PAR2 at E56↓T57, which removed the canonical tethered ligand and prevented trypsin activation. In HEK and KNRK cell lines and in nociceptive neurons of mouse dorsal root ganglia, Cat-S and a decapeptide mimicking the Cat-S-revealed tethered ligand-stimulated PAR2 coupling to Gαs and formation of cAMP. In contrast to trypsin, Cat-S did not mobilize intracellular Ca2+, activate ERK1/2, recruit β-arrestins, or induce PAR2 endocytosis. Cat-S caused PAR2-dependent activation of transient receptor potential vanilloid 4 (TRPV4) in Xenopus laevis oocytes, HEK cells and nociceptive neurons, and stimulated neuronal hyperexcitability by adenylyl cyclase and protein kinase A-dependent mechanisms. Intraplantar injection of Cat-S caused inflammation and hyperalgesia in mice that was attenuated by PAR2 or TRPV4 deletion and adenylyl cyclase inhibition. Cat-S and PAR2 antagonists suppressed formalin-induced inflammation and pain, which implicates endogenous Cat-S and PAR2 in inflammatory pain. Our results identify Cat-S as a biased agonist of PAR2 that causes PAR2- and TRPV4-dependent inflammation and pain. They expand the role of PAR2 as a mediator of protease-driven inflammatory pain.

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain.

Background: Proteases cleave protease-activated receptor-2 (PAR2), which activates transient receptor potential (TRP) ion channels to cause inflammation and pain. Results: Neutrophil elastase cleaves PAR2, resulting in Gαs-mediated cAMP formation, transient receptor potential vanilloid 4 (TRPV4) activation, and sensitization of nociceptive neurons, inflammation, and pain. Conclusion: Elastase causes PAR2- and TRPV4-mediated inflammation and pain. Significance: PARs and TRP channels mediate responses to diverse proteases. Proteases that cleave protease-activated receptor-2 (PAR2) at Arg36↓Ser37 reveal a tethered ligand that binds to the cleaved receptor. PAR2 activates transient receptor potential (TRP) channels of nociceptive neurons to induce neurogenic inflammation and pain. Although proteases that cleave PAR2 at non-canonical sites can trigger distinct signaling cascades, the functional importance of the PAR2-biased agonism is uncertain. We investigated whether neutrophil elastase, a biased agonist of PAR2, causes inflammation and pain by activating PAR2 and TRP vanilloid 4 (TRPV4). Elastase cleaved human PAR2 at Ala66↓Ser67 and Ser67↓Val68. Elastase stimulated PAR2-dependent cAMP accumulation and ERK1/2 activation, but not Ca2+ mobilization, in KNRK cells. Elastase induced PAR2 coupling to Gαs but not Gαq in HEK293 cells. Although elastase did not promote recruitment of G protein-coupled receptor kinase-2 (GRK2) or β-arrestin to PAR2, consistent with its inability to promote receptor endocytosis, elastase did stimulate GRK6 recruitment. Elastase caused PAR2-dependent sensitization of TRPV4 currents in Xenopus laevis oocytes by adenylyl cyclase- and protein kinase A (PKA)-dependent mechanisms. Elastase stimulated PAR2-dependent cAMP formation and ERK1/2 phosphorylation, and a PAR2- and TRPV4-mediated influx of extracellular Ca2+ in mouse nociceptors. Adenylyl cyclase and PKA-mediated elastase-induced activation of TRPV4 and hyperexcitability of nociceptors. Intraplantar injection of elastase to mice caused edema and mechanical hyperalgesia by PAR2- and TRPV4-mediated mechanisms. Thus, the elastase-biased agonism of PAR2 causes Gαs-dependent activation of adenylyl cyclase and PKA, which activates TRPV4 and sensitizes nociceptors to cause inflammation and pain. Our results identify a novel mechanism of elastase-induced activation of TRPV4 and expand the role of PAR2 as a mediator of protease-driven inflammation and pain.

Translational control by RGS2.

A regulator of G protein signaling, RGS2, moonlights in protein synthesis control.

Agonist-biased Trafficking of Somatostatin Receptor 2A in Enteric Neurons

Background: Somatostatin regulates gut function via neuronal somatostatin receptors. Results: Somatostatin susceptibility to degradation by endosomal endothelin-converting enzyme 1 (ECE-1) defines receptor function. Conclusion: ECE-1 regulates the duration of somatostatin receptor signaling and trafficking. Significance: Therapeutic somatostatin analogs are ECE-1-resistant, which underlies their prolonged actions. Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with β-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and β-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/β-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from β-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained β-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.

The Proline-rich N-terminal Domain of G18 Exhibits a Novel G Protein Regulatory Function

The protein G18 (also known as AGS4 or GPSM3) contains three conserved GoLoco/GPR domains in its central and C-terminal regions that bind to inactive Gαi, whereas the N-terminal region has not been previously characterized. We investigated whether this domain might itself regulate G protein activity by assessing the abilities of G18 and mutants thereof to modulate the nucleotide binding and hydrolytic properties of Gαi1 and Gαo. Surprisingly, in the presence of fluoroaluminate (AlF4−) both G proteins bound strongly to full-length G18 (G18wt) and to its isolated N-terminal domain (G18ΔC) but not to its GoLoco region (ΔNG18). Thus, it appears that its N-terminal domain promotes G18 binding to fluoroaluminate-activated Gαi/o. Neither G18wt nor any G18 mutant affected the GTPase activity of Gαi1 or Gαo. In contrast, complex effects were noted with respect to nucleotide binding. As inferred by the binding of [35S]GTPγS (guanosine 5′-O-[γ-thio]triphosphate) to Gαi1, the isolated GoLoco region as expected acted as a guanine nucleotide dissociation inhibitor, whereas the N-terminal region exhibited a previously unknown guanine nucleotide exchange factor effect on this G protein. On the other hand, the N terminus inhibited [35S]GTPγS binding to Gαo, albeit to a lesser extent than the effect of the GoLoco region on Gαi1. Taken together, our results identify the N-terminal region of G18 as a novel G protein-interacting domain that may have distinct regulatory effects within the Gi/o subfamily, and thus, it could potentially play a role in differentiating signals between these related G proteins.

The Ras‐binding domain region of RGS14 regulates its functional interactions with heterotrimeric G proteins

RGS14 is a 60 kDa protein that contains a regulator of G protein signaling (RGS) domain near its N‐terminus, a central region containing a pair of tandem Ras‐binding domains (RBD), and a GPSM (G protein signaling modulator) domain (a.k.a. Gi/o‐Loco binding [GoLoco] motif) near its C‐terminus. The RGS domain of RGS14 exhibits GTPase accelerating protein (GAP) activity toward Gαi/o proteins, while its GPSM domain acts as a guanine nucleotide dissociation inhibitor (GDI) on Gαi1 and Gαi3. In the current study, we investigate the contribution of different domains of RGS14 to its biochemical functions. Here we show that the full‐length protein has a greater GTPase activating activity but a weaker inhibition of nucleotide dissociation relative to its isolated RGS and GPSM regions, respectively. Our data suggest that these differences may be attributable to an inter‐domain interaction within RGS14 that promotes the activity of the RGS domain, but simultaneously inhibits the activity of the GPSM domain. The RBD region seems to play an essential role in this regulatory activity. Moreover, this region of RGS14 is also able to bind to members of the B/R4 subfamily of RGS proteins and enhance their effects on GPCR‐activated Gi/o proteins. Overall, our results suggest a mechanism wherein the RBD region associates with the RGS domain region, producing an intramolecular interaction within RGS14 that enhances the GTPase activating function of its RGS domain while disfavoring the negative effect of its GPSM domain on nucleotide dissociation. J. Cell. Biochem. 114: 1414–1423, 2013.

Fine-Tuning of GPCR Signals by Intracellular G Protein Modulators

Heterotrimeric G proteins convey receptor signals to intracellular effectors. Superimposed over the basic GPCR-G protein-effector scheme are three types of auxiliary proteins that also modulate Gα. Regulator of G protein signaling proteins and G protein signaling modifier proteins respectively promote GTPase activity and hinder GTP/GDP exchange to limit Gα activation. There are also diverse proteins that, like GPCRs, can promote nucleotide exchange and thus activation. Here we review the impact of these auxiliary proteins on GPCR signaling. Although their precise physiological functions are not yet clear, all of them can produce significant effects in experimental systems. These signaling changes are generally consistent with established effects on isolated Gα; however, the activation state of Gα is seldom verified and many such changes appear also to reflect the physical disruption of or indirect effects on interactions between Gα and its associated GPCR, Gβγ, and/or effector.

Resistance to age-related, normal body weight gain in RGS2 deficient mice

RGS2 (regulator of G protein signaling 2) is known to limit signals mediated via Gq- and Gs-coupled GPCRs (G protein coupled receptors), and it has been implicated in the differentiation of several cells types. The physiology of RGS2 knockout mice (rgs2(-/-)) has been studied in some detail, however, a metabolic phenotype has not previously been reported. We observed that old (21-24month) rgs2(-/-) mice weigh much less than wild-type C57BL/6 controls, and exhibit greatly reduced fat deposits, decreased serum lipids, and low leptin levels. Lower weight was evident as early as four weeks and continued throughout life. Younger adult male rgs2(-/-) mice (4-8months) were found to show similar strain-related differences as the aged animals, as well improved glucose clearance and insulin sensitivity, and enhanced beta-adrenergic and glucagon signaling in isolated hepatocytes. In addition, rgs2(-/-) pre-adipocytes had reduced levels of differentiation markers (Peroxisome proliferator-activated receptor γ (PPARγ); lipoprotein lipase (Lpl); CCAAT/enhancer binding protein α (CEBPα)) and also rgs2(-/-) white adipocytes were small relative to controls, suggesting altered adipogenesis. In wild-type animals, RGS2 mRNA was decreased in brown adipose tissue after cold exposure (7 h at 4 °C) but increased in white adipose tissue in response to a high fat diet, also suggesting a role in lipid storage. No differences between strains were detected with respect to food intake, energy expenditure, GPCR-stimulated lipolysis, or adaptive thermogenesis. In conclusion this study points to RGS2 as being an important regulatory factor in controlling body weight and adipose function.