Academic literature on the topic 'GPCR Signalling Pathways'

The discovery and extension of G-protein-coupled receptor (GPCR) transactivation-dependent signalling has enormously broadened the GPCR signalling paradigm. GPCRs can transactivate protein tyrosine kinase receptors (PTKRs) and serine/threonine kinase receptors (S/TKRs), notably the epidermal growth factor receptor (EGFR) and transforming growth factor-β type 1 receptor (TGFBR1), respectively. Initial comprehensive mechanistic studies suggest that these two transactivation pathways are distinct. Currently, there is a focus on GPCR inhibitors as drug targets, and they have proven to be efficacious in vascular diseases. With the broadening of GPCR transactivation signalling, it is therefore important from a therapeutic perspective to find a common transactivation pathway of EGFR and TGFBR1 that can be targeted to inhibit complex pathologies activated by the combined action of these receptors. Reactive oxygen species (ROS) are highly reactive molecules and they act as second messengers, thus modulating cellular signal transduction pathways. ROS are involved in different mechanisms of GPCR transactivation of EGFR. However, the role of ROS in GPCR transactivation of TGFBR1 has not yet been studied. In this review, we will discuss the involvement of ROS in GPCR transactivation-dependent signalling.

With >800 members, G protein-coupled receptors (GPCRs) are the largest class of cell-surface signalling proteins, and their activation mediates diverse physiological processes. GPCRs are ubiquitously distributed across all cell types, involved in many diseases and are major drug targets. However, GPCR drug discovery is still characterized by very high attrition rates. New avenues for GPCR drug discovery may be provided by a recent shift away from the traditional view of signal transduction as a simple chain of events initiated from the plasma membrane. It is now apparent that GPCR signalling is restricted to highly organized compartments within the cell, and that GPCRs activate distinct signalling pathways once internalized. A high-resolution understanding of how compartmentalized signalling is controlled will probably provide unique opportunities to selectively and therapeutically target GPCRs.

GPCRs (G-protein-coupled receptors) are members of a family of proteins which are generally regarded as the largest group of therapeutic drug targets. Ligands of GPCRs do not usually activate all cellular signalling pathways linked to a particular seven-transmembrane receptor in a uniform manner. The fundamental idea behind this concept is that each ligand has its own ability, while interacting with the receptor, to activate different signalling pathways (or a particular set of signalling pathways) and it is this concept which is known as biased signalling. The importance of biased signalling is that it may selectively activate biological responses to favour therapeutically beneficial signalling pathways and to avoid adverse effects. There are two levels of biased signalling. First, bias can arise from the ability of GPCRs to couple to a subset of the available G-protein subtypes: Gαs, Gαq/11, Gαi/o or Gα12/13. These subtypes produce the diverse effects of GPCRs by targeting different effectors. Secondly, biased GPCRs may differentially activate G-proteins or β-arrestins. β-Arrestins are ubiquitously expressed and function to terminate or inhibit classic G-protein signalling and initiate distinct β-arrestin-mediated signalling processes. The interplay of G-protein and β-arrestin signalling largely determines the cellular consequences of the administration of GPCR-targeted drugs. In the present review, we highlight the particular functionalities of biased signalling and discuss its biological effects subsequent to GPCR activation. We consider that biased signalling is potentially allowing a choice between signalling through ‘beneficial’ pathways and the avoidance of ‘harmful’ ones.

Heterotrimeric GPCRs (G-protein-coupled receptors) form the largest group of integral membrane receptor proteins and mediate diverse physiological processes. In addition to signalling via heterotrimeric G-proteins, GPCRs can also signal by interacting with various small G-proteins to regulate downstream effector pathways. The small G-protein superfamily is structurally classified into at least five families: the Ras, Rho/Rac/cdc42, Rab, Sar1/Arf and Ran families. They are monomeric G-proteins with molecular masses over the range 20–30 kDa, which function as molecular switches to control many eukaryotic cell functions. Several studies have provided evidence of crosstalk between GPCRs and small G-proteins. It is well documented that GPCR signalling through heterotrimeric G-proteins can lead to the activation of Ras and Rho GTPases. In addition, RhoA, Rabs, ARFs and ARF GEFs (guanine nucleotide-exchange factors) can associate directly with GPCRs, and GPCRs may also function as GEFs for small GTPases. In this review, we summarize the recent progress made in understanding the interaction between GPCRs and small GTPases, focusing on understanding how the association of small G-proteins with GPCRs and GPCR-regulatory proteins may influence GPCR signalling and intracellular trafficking.

The dynamic character of GPCRs (G-protein-coupled receptors) is essential to their function. However, the details of how ligands and signalling proteins stabilize a receptor conformation to trigger the activation of a given signalling pathway remain largely unexplored. Multiple data, including recent results obtained with the purified ghrelin receptor, suggest a model where ligand efficacy and functional selectivity are directly related to different receptor conformations. Importantly, distinct effector proteins (G-proteins and arrestins) as well as ligands are likely to affect the conformational landscape of GPCRs in different manners, as we show with the isolated ghrelin receptor. Such modulation of the GPCR conformational landscape by pharmacologically distinct ligands and effector proteins has major implications for the design of new drugs that activate specific signalling pathways.

Aims: G protein-coupled receptor (GPCR) transactivation of kinase receptors greatly expands the actions attributable to GPCRs. Thrombin, via its cognate GPCR, protease-activated receptor (PAR)-1, transactivates tyrosine and serine/threonine kinase receptors, specifically the epidermal growth factor receptor and transforming growth factor-β receptor, respectively. PAR-1 transactivation-dependent signalling leads to the modification of lipid-binding proteoglycans involved in the retention of lipids and the development of atherosclerosis. The mechanisms of GPCR transactivation of kinase receptors are distinct. We aimed to investigate the role of proximal G proteins in transactivation-dependent signalling. Main Methods: Using pharmacological and molecular approaches, we studied the role of the G⍺ subunits, G⍺q and G⍺11, in the context of PAR-1 transactivation-dependent signalling leading to proteoglycan modifications. Key Findings: Pan G⍺q subunit inhibitor UBO-QIC/FR900359 inhibited PAR-1 transactivation of kinase receptors and proteoglycans modification. The G⍺q/11 inhibitor YM254890 did not affect PAR-1 transactivation pathways. Molecular approaches revealed that of the two highly homogenous G⍺q members, G⍺q and G⍺11, only the G⍺q was involved in regulating PAR-1 mediated proteoglycan modification. Although G⍺q and G⍺11 share approximately 90% homology at the protein level, we show that the two isoforms exhibit different functional roles. Significance: Our findings may be extrapolated to other GPCRs involved in vascular pathology and highlight the need for novel pharmacological tools to assess the role of G proteins in GPCR signalling to expand the preeminent position of GPCRs in human therapeutics.

The calcium-sensing receptor (CASR) is a class C G-protein-coupled receptor (GPCR) that detects extracellular calcium concentrations, and modulates parathyroid hormone secretion and urinary calcium excretion to maintain calcium homeostasis. The CASR utilises multiple heterotrimeric G-proteins to mediate signalling effects including activation of intracellular calcium release; mitogen-activated protein kinase (MAPK) pathways; membrane ruffling; and inhibition of cAMP production. By studying germline mutations in the CASR and proteins within its signalling pathway that cause hyper- and hypocalcaemic disorders, novel mechanisms governing GPCR signalling and trafficking have been elucidated. This review focusses on two recently described pathways that provide novel insights into CASR signalling and trafficking mechanisms. The first, identified by studying a CASR gain-of-function mutation that causes autosomal dominant hypocalcaemia (ADH), demonstrated a structural motif located between the third transmembrane domain and the second extracellular loop of the CASR that mediates biased signalling by activating a novel β-arrestin-mediated G-protein-independent pathway. The second, in which the mechanism by which adaptor protein-2 σ-subunit (AP2σ) mutations cause familial hypocalciuric hypercalcaemia (FHH) was investigated, demonstrated that AP2σ mutations impair CASR internalisation and reduce multiple CASR-mediated signalling pathways. Furthermore, these studies showed that the CASR can signal from the cell surface using multiple G-protein pathways, whilst sustained signalling is mediated only by the Gq/11 pathway. Thus, studies of FHH- and ADH-associated mutations have revealed novel steps by which CASR mediates signalling and compartmental bias, and these pathways could provide new targets for therapies for patients with calcaemic disorders.

Abstract The epidermal growth factor receptor (EGFR) plays a key role in the regulation of important cellular processes under normal and pathophysiological conditions such as cancer. In human mammary carcinomas the EGFR is involved in regulating cell growth, survival, migration and metastasis and its activation correlates with the lack of response in hormone therapy. Here, we demonstrate in oestrogen receptor-positive and -negative human breast cancer cells and primary mammary epithelial cells a cross-communication between G protein-coupled receptors (GPCRs) and the EGFR. We present evidence that specific inhibition of ADAM15 or TACE blocks GPCR-induced and proHB-EGF-mediated EGFR tyrosine phosphorylation, downstream mitogenic signalling and cell migration. Notably, activation of the PI3K downstream mediator PKB/Akt by GPCR ligands involves the activity of sphingosine kinase (SPHK) and is independent of EGFR signal transactivation. We conclude that GPCR-induced chemotaxis of breast cancer cells is mediated by EGFR-dependent and -independent signalling pathways, with both parallel pathways having to act in concert to achieve a complete migratory response.

The EGFR (epidermal growth factor receptor) plays a key role in the regulation of essential normal cellular processes and in the pathophysiology of hyperproliferative diseases such as cancer. Recent investigations have demonstrated that GPCRs (G-protein-coupled receptors) are able to utilize the EGFR as a downstream signalling partner in the generation of mitogenic signals. This cross-talk mechanism combines the broad diversity of GPCRs with the signalling capacities of the EGFR and has emerged as a general concept in a multitude of cell types. The molecular mechanisms of EGFR signal transactivation involve processing of transmembrane growth factor precursors by metalloproteases which have been recently identified as members of the ADAM (adisintegrin and metalloprotease) family of zinc-dependent proteases. Subsequently, the EGFR transmits signals to prominent downstream pathways, such as mitogen-activated protein kinases, the phosphoinositide 3-kinase/Akt pathway and modulation of ion channels. Analysis of GPCR-induced EGFR activation in more than 60 human carcinoma cell lines derived from different tissues has demonstrated the broad relevance of this signalling mechanism in cancer. Moreover, EGFR signal transactivation was linked to diverse biological processes in human cancer cells, such as cell proliferation, migration and anti-apoptosis. Together with investigations revealing the importance of this GPCR–EGFR cross-talk mechanism in cardiac hypertrophy, Helicobacter pylori-induced pathophysiological processes and cystic fibrosis, these findings support an important role for GPCR ligand-dependent EGFR signal transactivation in diverse pathophysiological disorders.

Alteration in [Ca2+]i (the intracellular concentration of Ca2+) is a key regulator of many cellular processes. To allow precise regulation of [Ca2+]i and a diversity of signalling by this ion, cells possess many mechanisms by which they are able to control [Ca2+]i both globally and at the subcellular level. Among these are many members of the superfamily of GPCRs (G-protein-coupled receptors), which are characterized by the presence of seven transmembrane domains. Typically, those receptors able to activate PLC (phospholipase C) enzymes cause release of Ca2+ from intracellular stores and influence Ca2+ entry across the plasma membrane. It has been well documented that Ca2+ signalling by one type of GPCR can be influenced by stimulation of a different type of GPCR. Indeed, many studies have demonstrated heterologous desensitization between two different PLC-coupled GPCRs. This is not surprising, given our current understanding of negative-feedback regulation and the likely shared components of the signalling pathway. However, there are also many documented examples of interactions between GPCRs, often coupling preferentially to different signalling pathways, which result in a potentiation of Ca2+ signalling. Such interactions have important implications for both the control of cell function and the interpretation of in vitro cell-based assays. However, there is currently no single mechanism that adequately accounts for all examples of this type of cross-talk. Indeed, many studies either have not addressed this issue or have been unable to determine the mechanism(s) involved. This review seeks to explore a range of possible mechanisms to convey their potential diversity and to provide a basis for further experimental investigation.

L’obesità è una condizione patologica per la quale non è stato ancora possibile identificare un trattamento farmacologico efficace. Il controllo dell’appetito è regolato da diversi fattori, la cui integrazione da parte dell’ipotalamo risulta nella generazione di specifiche risposte che regolano il bilancio energetico. TLQP-21 è un neuro-peptide coinvolto nella regolazione di diverse funzioni fisiologiche, incluso metabolismo, diabete, dolore e funzioni gastriche. Nonostante la recente identificazione di due potenziali recettori per TLQP-21, ad oggi, il meccanismo d’azione con cui agisce questo peptide rimane largamente sconosciuto. TLQP-21 viene attualmente proposto come un nuovo target per la cura di diverse patologie. Pertanto, lo scopo del nostro studio è stato quello di studiare il pathway intracellulare attivato dal legame di TLQP-21 col suo recettore, al fine di creare in futuro nuovi modelli utili allo sviluppo di farmaci che agiscano su questo sistema. Abbiamo inizialmente caratterizzato la capacità di TLQP-21 di indurre, in maniera dose-dipendente, un aumento di Ca2+ intracellulare nelle cellule CHO, N9, e RAW264.7. In particolare, l’aumento di Ca2+ intracellulare osservato dopo stimolazione con TLQP-21 è dovuto al rilascio di Ca2+ dal reticolo endoplasmatico, come dimostrato dalla capacità della tapsigargina di inibire l’effetto di TLQP-21. È noto che il rilascio di Ca2+ dal reticolo endoplasmatico è regolato dall’attivazione della PLC e dalla conseguente produzione di IP3 che si lega a specifici recettori presenti sul reticolo endoplasmatico. Nelle cellule CHO, N9 e RAW264.7 il trattamento con l’inibitore della PLC U73122 e con l’antagonista dei recettori IP3 2-APB riduce l’attività di TLQP-21, confermando per questo peptide un meccanismo d’azione PLC-dipendente. Inoltre, nelle cellule CHO, TLQP-21 induce una rapida defosforilazione della PLCγ1, suggerendo che l’aumento di Ca2+ osservato in seguito a stimolazione con TLQP-21 sia mediato dal legame del peptide con un recettore accoppiato a una proteina di tipo Gq, che a sua volta attiverebbe la PLCβ. Il rilascio di Ca2+ dal reticolo indotto da TLQP-21 attiva inoltre un flusso di Ca2+ dall’ambiente extracellulare, come dimostrato dal trattamento con SKF-96365 e YM-58483, due specifici inibitori dello store-operated calcium entry process. Nelle cellule CHO, TLQP-21 induce un aumento della fosforilazione della PKC e, di conseguenza, di ERK1/2. Inoltre, l’aumento di Ca2+ intracellulare indotto da TLQP-21 stimola l’attivazione di Akt/PKB. I risultati di questa ricerca suggeriscono quindi che il recettore stimolato da TLQP-21 appartenga alla famiglia dei recettori accoppiati a proteine Gq. Il legame di TLQP-21 con questo recettore, attivando la PLC, stimola la produzione di secondi messaggeri che a loro volta inducono il rilascio di Ca2+ dal reticolo endoplasmatico e il conseguente flusso in entrata di Ca2+ dall’ambiente extracellulare. In conclusione, la nostra ricerca fornisce nuove evidenze riguardo il meccanismo di azione di TLQP-21, e fornisce nuove indicazioni sul recettore specifico espresso in alcune linee cellulari che rispondono a TLQP-21. La futura caratterizzazione molecolare di tale recettore potrà permettere lo sviluppo di modelli sperimentali utili per la ricerca di farmaci per il trattamento di diverse patologie, inclusi obesità e diabete.
Obesity is a global epidemic for which the current weight loss therapies are relatively ineffective. Many central and peripheral factors are involved in the mechanisms controlling eating behaviour, and the integration of these signals within the hypothalamus results in the generation of specific responses aimed at regulating energy balance. TLQP-21 is a novel neuropeptide that has been implicated in the regulation of energy homeostasis, nociception, gastric function and several other physiologic functions. Although recent studies identified different receptors as the targets for TLQP-21, its molecular mechanisms of action at the cellular level remain largely unknown. Thus, since TLQP-21 is emerging as a novel target for obesity-associated disorders, diabetes, neuropathic pain, and other human pathologies, the purpose of this study was to better investigate the intracellular signalling pathway activated by the peptide-receptor interaction. Here, using intracellular calcium mobilization assay and western blot analysis, we have pharmacologically characterized the intracellular signalling pathway activated by TLQP-21 in ovary, macrophage and microglial cells. TLQP-21 dose-dependently stimulated a rapid and transient intracellular Ca2+ increase in CHO, RAW264.7 and N9 cells, and repeated exposure to the peptide resulted in a reduced response, indicating a possible desensitization mechanism of TLQP-21 receptor. In particular, TLQP-21 stimulation induced an increase of cytoplasmic Ca2+ levels that was sustained by Ca2+ release from the ER, since treatment of the cells with thaspigargin reduced the TLQP-21-mediated increase of intracellular Ca2+. The release of Ca2+ from the ER store is regulated by the activation of PLCs and the subsequent production of IP3 that binds to its receptors on the surface of the ER. In our cellular systems, TLQP-21 activity was reduced by the treatment with the PLC inhibitor U73122 and the IP3R antagonist 2-APB, confirming a PLC-dependent mechanism of action for the peptide. Furthermore, TLQP-21 induced a rapid dephosphorylation of PLCγ1 in CHO cells, suggesting that Ca2+ response to TLQP-21 is mediated by the binding of the peptide to a Gq-coupled receptor that in turn activates PLCβ. Ca2+ release from the ER activated Ca2+ entry from the extracellular environment, as demonstrated by the treatment of the cells with SKF-96365 and YM-58483, two specific inhibitors of the SOCE pathway. In CHO cells, TLQP-21 induced also an increase of PKC phosphorylation and, afterwards, of ERK1/2 phosphorylation. Moreover, the increase of cytosolic Ca2+ concentration following TLQP-21 administration, stimulated the activation of Akt/PKB. Our results suggest that the receptor stimulated by TLQP-21 belongs to the family of the Gq-coupled receptors, that activates membrane-lipid derived second messengers which thereby induce Ca2+ mobilization from the ER followed by a slower store-operated Ca2+ entry from outside the cell. In conclusion, our research provides additional evidences about the molecular mechanisms of action of TLQP-21, and could be useful to open new approaches to improve the treatment of several human disorders, including obesity and diabetes.

The Hedgehog signalling pathway is an essential developmental pathway present in all bilaterians that is involved in embryogenesis, body patterning and stem cell homeostasis. Dysregulation of the Hh pathway leads to various kinds of cancer, such as basal cell carcinoma and medulloblastoma. Smoothened (SMO), a Frizzled-type G-protein coupled receptor (GPCR), is the essential transmembrane signal transducer within the Hh pathway, conveying the signal from the upstream transmembrane protein, Patched1 (Ptc1), to the downstream intracellular proteins. The mechanisms by which SMO transmits the Hh signal from the extracellular environment, through the plasma membrane and to the intracellular proteins are not known. In this thesis, I present my work into the structural and functional characterisation of the extracellular and transmembrane domains (TMD) of human SMO in order to better understand the molecular mechanisms of its signal transduction. The extracellular region of SMO contains a highly conserved cysteine-rich domain (CRD) and a linker domain (LD). I present the first crystal structure of the CRD, LD and TMD of SMO, which is also the first crystal structure of a GPCR with a large functional extracellular domain. This structure revealed a domain architecture for SMO that enables regulation of its transmembrane domain by its extracellular domains. It also revealed a cholesterol molecule bound to the CRD, which we subsequently determined to be a new endogenous small-molecule agonist for SMO. I present five further structures of SMO bound to different small molecule agonists and antagonists. Together, these structures demonstrate that the position of the CRD relative to the TMD reflects the activation state of SMO. We also generated nanobodies against the extracellular region of SMO in order to stabilise its conformation. These studies not only improve our understanding of the workings of a key transmembrane protein within a fundamental signalling pathway but will also aid efforts to develop better therapeutics for an important cancer target.

Signal transduction is a mode of cellular communication essential for an organism’s sustenance and survival. Cell communication in higher organisms is largely executed by two classes of cell surface receptors i.e. G-protein coupled Receptors (GPCRs) and receptor tyrosine kinases (RTKs). Though the activation of each of these receptors have shown to regulate cellular responses through a linear pathway the underlying signaling events are much more complicated. A number of studies have shown that the signaling regulators of the RTK mediated pathway can be cross-activated by GPCRs activation via a process termed as ‘transactivation.’ However, these studies are limited, as they have largely missed the spatio-temporal dynamics of the signaling regulators involved in the transactivation pathway. With this premise, the present research was structured to investigate the spatiotemporal dynamics of MAPK cascade transactivated by GPCR signalling pathways in live cells. Towards this broad objective, we subdivided the study into three part

In the work described in this thesis, analytical methods for the detection and quantification of peptide hormones featuring on-line analyte concentration, post-separation tagging and HPLC-fluorescence detection were presented. These methods were used to detect and quantify calcitonin (CT) and its prohormones glycyllysyllysine-extended CT (CTGKK), glycyllysine-extended CT (CTGK) and glycine-extended CT (CT-G) for the first time, in DMS53 small cell lung carcinoma (SCLC) cell culture medium and lysate. Additionally, novel glycosylated versions of each species were also identified, suggesting the presence of a parallel biosynthetic pathway in DMS53. Extracellular but not intracellular levels of CT were reduced as a result of treatment with biosynthesis inhibitors, and it was suggested CT precursor flux through the glycosylated pathway acts as a bypass mechanism to maintain intracellular CT levels. Moreover, the up-regulation of extracellular levels of CT-related species in response to increased medium volume provided evidence of a homeostatic feedback loop maintaining extracellular CT concentrations. To interrogate the mechanism of this feedback, DMS53 cultures were treated with a specific human calcitonin receptor (hCTR) agonist, SUNB8155, to determine if the hCTR is involved in the regulation of CT. It was observed that the relative levels of extracellular CT increased with SUNB8155 treatment, but that the relative levels of the intracellular CT-related species were unchanged. This suggested that hCTR is expressed in DMS53, and that activation of the receptor influences the expression and biosynthetic processing of CT-related species. To investigate this hypothesis, hCTR was identified in DMS53 cells using reverse transcription PCR and Western blot analyses. Specifically, transcriptional and translational evidence of the isoform hCTR2 was identified. Thus, for the first time,hCTR activation was implicated in the up-regulation of CT. This suggested that a positive autocrine feedback loop was operating in DMS53, and based on the hCTR2 isoform, may be mediated by signal transduction through the cAMP- and Ca2+- dependent signalling pathways. To assess which signalling enzymes are activated by hCTR, signal transduction pathways were investigated using small molecule enzymes inhibitors, and their effects on the levels of CT-related species observed. It was observed that treatment of DMS53 cultures with the protein kinase C inhibitor, GF109203X had an effect on the levels of CT-related species in the medium. Again, the relative levels of the intracellular CT-related species were not changed by treatment with this inhibitor. This implicated PKC as a component of the hCTR signal transduction pathway. It was concluded that DMS53 cultures have mechanisms to maintain the intracellular and extracellular concentrations of CT-related species. The concentration of extracellular CT is regulated by a positive feedback mechanism, mediated by hCTR activation, and subsequent signalling involving PKC and AC. Treatment with biosynthetic and signalling inhibitors had no significant effect on the intracellular levels of CT-related species, demonstrating that DMS53 cultures prioritise tight control of intracellular concentrations over extracellular concentrations. With the methodology to detect and quantify peptide hormones in cell culture medium and lysate in hand, the generality of CT glycosylation was explored. Preliminary experiments successfully characterised the presence of glycosylated CT and CT-G in the medullary thyroid carcinoma cell line, TT. To broaden the range of detected hormones, HPLC-fluorescence methodology was developed to detect and quantify oxytocin (OT) and its precursors, and this methodology was used to investigate the presence of OT in the DMS79 SCLC cell line.