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.
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.
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.
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.
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.