Physiology and Pharmacology

Research

• Cardiovascular Research
• Cell Biology Research
  • Cell Biology Research Groups
  • Cell Signalling
    • Graeme Henderson
    • Ingerborg Hers
    • Eamonn Kelly
    • Stuart Mundell
    • Alastair Poole
  • Cell migration and repair
  • CFTR Regulation
• Neuroscience Research
• Teaching Development Research Group
• All Research Groups

 

External Groups

• AIMS Centre/CETL
• Bristol Heart Institute
• Bristol Neuroscience
• Microvascular Research Laboratories (MVRL)
• MRC Centre for Synaptic Plasticity

Cell Signalling

Signalling in GPCRs | Signalling in platelets | Action of PKC isoforms in platelet signalling

Cells respond to their external environments via the interactions of small molecules with receptor proteins on the cell surface, initiating signalling cascades that alter the functions of numerous other proteins. Changes in cellular motility, membrane excitability and gene expression can all be profoundly changed by receptor activation or inhibition, as well as by how many receptors are expressed in a particular cell. Thus the control of the signalling cascades is fundamental to the control of cellular function.

G-Protein coupled receptors

One of the receptor families that is heavily studied in the School of Physiology and Pharmacology is the G-protein coupled receptors, GPCRs. More than 800 GCPRs are represented in the human genome and they perform a myriad of functions, responding to hormones, modulating synaptic responses or controlling Ca2+ bursts, among many other functions, via the activation of either the α or βγ subunits of G-proteins.

There are a six different classes of GPCRs: research within the school focuses on two of those classes - rhodopsin-like receptors (class A) and the metabotropic glutamate receptors (class C). These two types of GPCR are structurally distinct. While both have a 7-transmembrane domain motif, the class C receptors have a large N-terminus containing the ligand binding domain and class A receptors bind ligands in a pocket formed by the packed transmembrane domains. However, much of the control of their expression, trafficking and functioning is common across many different receptor subtypes and thus points to general regulatory mechanisms that act across multiple cell types.

Trafficking of GPCRs

A major focus of research is into the mechanisms of GPCR desensitisation and recycling - trafficking. Such desensitisation is of the common features of GPCR functioning, and indeed receptor functioning in general, where continued exposure of the receptor to agonist results in a reduction or total loss of activity. There are multiple ways in which GPCR activity can be reduced - reducing affinity of agonist, preventing G-protein re-binding to the receptor or by reducing the number of receptors expressed at the exposed cell surface. Please note that down-regulation refers to the lysolytic degradation of internalised receptor rather than to the internalisation process itself.

Desensitisation - the classical view ....

In the classical view of GPCR desensitisation, the agonist-bound receptor is phosphorylated at ser/thr residues in the 3rd intracellular loop or C-terminal domain by a relevant G-protein receptor kinase (GRK). This leads to the binding of β-arrestin that in turn blocks the re-association of the G-protein with the receptor, thus preventing further activation. Binding of β-arrestin induces clathrin-mediated internalisation, where the agonist dissociates from the receptor, leading to de-phosphorylation and the return of the receptor to the cell surface where it re-associates with G-protein - re-sensitisation (detailed in Kelly et al 2008). Sorting nexins (SNX-BAR proteins) are also involved in both clathrin- and non clathrin-mediated endocytosis (reviewed in van Weering et al 2010).  They  associate with another protein complex, the retromer, in order to transport endocytic vesicles to the trans-golgi network and tether them there.

... but it doesn't always work like that ...

The rate at which each step during GPCR desensitisation occurs can vary from receptor to receptor, even those that are closely related. For instance, the A1 adenosine receptor internalises at a much slower rate than other members of the adenosine receptor family (Mundell & Kelly, 2010). Indeed while the classical view may form the basis of the internalisation mechanism for many GPCRs, many subtle (and not so subtle) variations exist. Thus we have recently shown that the adenosine A2B receptor can undergo both GRK/arrestin-dependent and independent desensitisation and have suggested that the binding of β-arrestin need not be dependent on GRK phosphorylation (Mundell et al 2010).

Other work has suggested that purinergic receptor recycling in platelets also does not necessarily follow the classical trafficking pathway and that SNX1 may play multiple roles in endocytic trafficking of GPCRs. Thus,  P2Y(1) and Py2(12) receptors, which are expressed in platelets, are recycled via different pathways; PY2(1)  receptors follow a slow, SNX1-dependent pathway, while P2Y(12) receptors recycle quickly (Nisar et al 2010). si-RNA-mediated knockdown of SNX1  increased surface expression of P2Y(1) but had no effect on agonist-mediated degradation. In addition, inhibition of proteins known to be present in the retromer had no effect on P2Y(1) trafficking. Thus SNX-dependent control of P2Y(1) recycling is independent of the retromer.

Signalling in platelets

Platelet activation following damage to a blood vessel involves the co-ordinated stimulation of multiple, interlocking signalling cascades, many of which involve the activation of PKC. Another pathway we have been investigating is signalling via Akt, a ser/thr kinase classically involved in cell survival, proliferation and metabolism. In addition, it has also been shown to be involved in neuronal plasticity and it plays a contributary role in platelet function.

Little is known about how Akt is regulated in platelets. Using mouse models, it has been shown to be doubly phosphorylated - on S473 by mTORC2 and on T308 by PDK1 but the relative abundance of Akt isoforms in mice and humans are different, with Akt1predominating ion mice while Akt2 is more prevalent in humans. We have thus investigated the phosphorylation of Akt in human platelets and have shown that mTORC2-dependent phosphorylation of S473 is not required to activate Akt1 and that it is independent of phosphorylation of T308 (Moore et al 2011). Furthermore, PRAS40 was identified as an Akt2 substrate in platelets.

Role of PKC isoforms in platelet function and thrombosis

PKC and thrombosis

The protein kinase C (PKC) family is a major regulator of platelet granule secretion, integrin activation, aggregation, spreading and procoagulant activity. Different PKC isoforms are important in different, often opposing, functions (reviewed in Harper & Poole 2010a). Thus PKCα and PKCβ positively regulate thrombus formation but play no role in cell adhesion while PKCθ, a novel PKC isoform, negatively regulates thrombus formation and has a small positive effect on cell adhesion (Hall et al 2008; Konopatskaya et al 2009). The different roles played by the various isoforms in thrombus formation has also been confirmed by testing  them side-by-side under standardized conditions (Gilio et al 2010).

PKC in Ca2+ signaling

We have also been investigating the roles played by PKC isoforms in Ca2+ handling within platelets. Agonists increase intracellular Ca2+ concentrations either by opening plasma membrane bound channels or via purinergic receptor-mediated release of Ca2+ from intracellular stores. A major component of Ca2+ handling combines these two mechanisms - lowering of Ca2+ held in stores results in the activation of plasma membrane localised channels in a process known as store-operated calcium entry, SOCE. We have recently shown that PKCα positively regulates SOCE, but not by direct interaction with either Ca2+ stores or store-operated channels (SOCs). Rather regulation is indirect, via the Ca2+/Na+ exchanger (CNX - Harper et al 2010). SOCE was reduced following PKC inhibition and in PKCα-/- mice. Such inhibtion was unaffected by blockade of either purinergic receptors and broad spectrum cation channels, but was replicated by blockade of the CNX.

In contrast to PKCα, PKCθ negatively regulates intracellular Ca2+ concentration. Regulation is by two separate mechanisms - inhibition of both store-independent Ca2+ entry and purinergic receptor-dependent release of Ca2+ from stores (Harper & Poole 2010b). Activation of GPVI results in a two stage increase in intracellular Ca2+. A rapid, short-lived increase in Ca2+ concentration represents the opening of membrane channels (peak increase) that is both enhanced in PKCθ-/- mice and is sensitive to purinergic antagonists. A more sustained increase in Ca2+ concentration represents the release of Ca2+ from stores. This sustained increase is also enhanced in PKCθ-/- mice and but is unaffected by purinergic receptor antagonists. However, it does not repesent an increase in Ca2+ release, but enhanced store-independent Ca2+ entry via non-selective cation channels as PKCθ does not directly regulate Ca2+ release from stores (Harper & Poole 2010b).

Thus, PKCθ negatively regulates ADP secretion and, indirectly, SOCE as well as restricting store-independent Ca2+ entry during GPVI signalling. It would also act as a 'brake' on SOCE.