Biol220 – Cellular Signalling Inositol Phospholipid Turnover and Ca2+ Signalling Calcium (Ca2+) as a signalling molecule Extracellular signals often cause a transient rise in the cytosolic [Ca2+]. In certain cells (e.g. neurones) the Ca2+ originates in the extracellular fluid; however, in many cells, the absence of Ca2+ in the extracellular fluid does not inhibit numerous Ca2+-mediated processes. Extracellular stimuli can provoke the release of Ca2+ from intracellular reservoirs (e.g. endoplasmic reticulum). This Ca2+ release must be mediated by an Visualization of Ca2+ in intracellular signal – cyclic nucleotides are not zebrafish embryos by involved. injecting them with aequorin; a photoprotein from the luminescent There is a correlation between mobilization of jellyfish that reacts with 2+ intracellular Ca and the turnover of Ca2+ and emits blue light phosphatidylinositol-4,5-bisphosphate, a minor at ~460 nm. component of the plasma membrane. Ca2+ signalling – the basics. The Phosphoinositide (PI) Signalling Pathway More than 25 different cell-surface receptors utilize the phosphoinositide (PI) signalling pathway. Adrenaline acting at 1-receptors, vasopressin acting at V1 receptors, and ADP and ATP acting at P2 receptors, all utilize this pathway to stimulate glycogen breakdown in the liver. Acetylcholine, acting through the PI pathway, stimulates amylase secretion from the pancreas. Thrombin stimulates aggregation of platelets through this pathway. Phospholipase C catalyzes the hydrolysis of PIP2 This reaction takes place in the plasma membrane and involves the breakdown of constituent phospholipids of the plasma membrane lipid bilayer. Between 2 and 8 % of the lipids of eukaryotic membranes are inositol-containing lipids. The three main forms are phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5- bisphosphate (PIP2). These are collectively known as phosphoinositides. Although the least plentiful, the most important of these is PIP2. PIP2 is cleaved by a phosphoinositide-specific phospholipase C to generate 2 products that have second messenger roles: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Phospholipase C 10 isoforms of mammalian phospholipase C (PLC) have been identified. They are all single polypeptides and can be divided into three types, , and , on the basis of size and amino acid sequence. Only the -isoform, with the G-protein-binding domain can be stimulated directly by G-proteins. There are two distinct mechanisms linking receptor occupancy to phospholipase C activation : PLC- isozymes are activated by the heterotrimeric G protein Gq. PLC- isozymes are activated by tyrosine phosphorylation. This may be catalyzed by a tyrosine kinase-linked receptor, e.g. EGF, PDGF, FGF or NGF receptors. The association of PLC- with the membrane-bound receptor brings this otherwise cytosolic enzyme, into contact with its substrate. At the amino terminus of the phospholipase C polypeptide is a pleckstrin homology domain (PH). This 120 residue domain binds a lipid head group such as that of PIP2. The PH and C2 domains help to position the enzyme’s catalytic site for ready access to the phosphodiester bond of the membrane lipid substrate, PIP2. IP3 and DAG as secondary messengers IP3 is a small, water-soluble molecule that leaves the plasma membrane and diffuses rapidly through the cytosol. It releases Ca2+ from the 2+ endoplasmic reticulum (ER) by binding to IP3-gated Ca channels (IP3 receptors) in the ER membrane. DAG is a lipophilic compound and remains associated with the plasma membrane. It performs two roles as a second messenger: 2+ 1. It activates protein kinase C. The initial rise in [Ca ]i induced by IP3 causes protein kinase C to translocate from the cytosol to the plasma membrane. Here it is activated by Ca2+, DAG and phosphatidylserine. Activated protein kinase C can phosphorylate, and thereby modulate the activity, of a range of proteins (inc. glycogen synthase, smooth muscle myosin light chains). 2. It can be cleaved to release arachidonic acid, which is a messenger in its own right (used in the biosynthesis of prostaglandins, prostacyclins, thromboxanes and leukotrienes). DAG, like IP3, acts transiently because it is rapidly metabolized. It can be phosphorylated to phosphatidate or it can be hydrolyzed to glycerol and its constituent fatty acids. Inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) recycling. Calmodulin Calmodulin is a small (17 kDa) Ca2+- binding protein that serves as a Ca2+ sensor in nearly all eukaryotic cells. It consists of 2 globular regions connected by a long, flexible helical region. Each of the globular domains can bind 2 Ca2+. Calmodulin is activated by binding 3 or 4 Ca2+. Ca2+ binding leads to a major conformational change in calmodulin, enabling it to interact with target proteins. Ca2+/calmodulin is responsible for activating a variety of enzymes involved in intracellular signalling, including CAM kinase II, calcineurin, adenylyl cyclase type I, nitric oxide synthase and PDE-I. The PI Signalling Pathway. The secondary messenger functions of IP3 and DAG explain many cellular phenomena. Nerve impulses mobilize Ca2+ in skeletal muscle by releasing neurotransmitters that bind to PLC-activating receptors. This results in the 2+ generation of IP3 which mobilizes Ca from the sarcoplasmic reticulum and triggers muscle contraction. The enzyme that is responsible for the hydrolysis of inositol-1-phosphate + (itself a breakdown product of IP3) is inhibited by Li . The therapeutic usefulness of Li+ in controlling mood swings in manic-depressives suggests that this illness is caused by a defect in the PI signalling system in the brain. Several polypeptide growth factors mobilize IP3 and DAG, which in turn stimulate cell proliferation. The v-sis oncogene (which specifies an analogue of PDGF) may act by permanently switching on the PI signalling system, forcing cells into a state of continuous proliferation. Phorbol esters (e.g. 12-O-tetra-decanoylphorbol-13-acetate) are potent activators of protein kinase C and are very effective tumour promoters (i.e. they increase the potency of known carcinogens). Significantly, Phorbol esters induce the biosynthesis of the transcription factor AP-1. 2+ [Ca ]i oscillations Stimulating single hepatocytes with a low physiological dose of an agonist induces, not a sustained rise in Ca2+ concentration, but a series of discrete, repetitive oscillations in cytosolic Ca2+ concentration. 2+ [Ca ]i oscillations have been recorded in many different cell types, and can now be regarded as a nearly universal mode of signalling in both excitable and non-excitable cells. Summary Extracellular signals often cause a transient rise in the cytosolic [Ca2+]. This Ca2+ originates from intracellular stores and its release is correlated with plasma membrane PI turnover. Phospholipase C catalyzes the hydrolysis of PIP2 to DAG and IP3 – both of which have secondary messenger functions. 2+ DAG is an activator of PK-C and IP3 activates Ca release from intracellular stores. The signalling activity of Ca2+ is usually mediated by Ca2+ binding proteins such as calmodulin. Ca2+calmodulin can activate the calmodulin-dependent protein kinases. Ca2+ signalling shows oscillatory or wave-like behaviour in intact cells..