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IP3-Signalling By: Jonathan Pan, Pranjal Bhatt, Mark Ng, Mohammad Alkhalili Course: PHM 142 Metabolic Biochemistry and Immunolgy PHM142 Fall 2012 Coordinator: Dr. Jeffrey Henderson Instructor: Dr. David Hampson
What is IP3 ? • Second messenger activated by ligand binding to a g-protein coupled receptor • Product of Phospholipid hydrolysis • Causes the release of Calcium from ER
Function • Activates cell proliferation -> Growth Factor signalling eg. EGF and FGF • Muscle contraction -> Release of Calcium can cause Smooth muscle contraction • Higher order inositol phosphate in immune cells
IP3- Signaling Pathway • second messenger that acts on a receptor to increase to intracellular Ca+2 • Ca+2 signalling important in a wide variety of physiological roles • two essential pathways that generate IP3 • First pathway initiated by PLC (Phospholipase-C) • Second pathway initiated by PI3K
Phospholipase C • Class of enzymes that cleave phospholipid just before the phosphate group • Phospoinostide-specific phospholipase C that cleaves PIP2 into IP3 and DAG (diacyl glycerol) • The most common pathway that increases cytoplasmic calcium concentration
IP3 binding and Ca+2 release • IP3 accumulates rapidly and transiently • Binds to its intracellular receptor, IP3R located on intracellular stores of calcium, eg. Endoplasmic Reticulum • Binding of IP3 to the IP3R receptor results in Ca+2 release from the ER lumen to the cytoplasm
IP3 Receptor overview • IP3R’s are tetramers that act as ligand gated channels facilitating the release of Ca2+ from the ER • Note that they are not just restricted to the ER, but have also been shown to mediate the release of Ca2+ from other intracellular organelles such as the nuclear envelope, Golgi and secretory vesicles
IP3 Receptor structure • IP3R’s are comprised of 4 large subunits – 2700 residues each and each subunit contains a single IP3 binding site. • Primary structure of IP3R contains 3 domains: • An IP3 binding domain near the N terminus • A coupling domain in the middle • A transmembrane spanning domain near the C terminus that anchors the protein to the membrane
IP3 Receptor • Shows positive cooperativity, suggesting several subunits (perhaps all 4) must bind IP3 before the channel opens. • The opening of these channels is regulated not only by IP3 but Ca2+ itself, the details are not fully understood yet but we will go over the main theory behind how this is thought to work
IP3 Activation • As IP3 binds it causes a conformational change in the receptor which reveals a Ca2+ binding site. • Binding of Ca2+ to this newly revealed site allows the suppressor domain to dislodge itself from the gatekeeper. • This allows the gatekeeper to relax and allow ions to access the pore.
IP3 Inhibition • Ca2+ can also act to inhibit opening of the channel in the absence of IP3. The suppressor region contains a lobe which also has a Ca2+ binding site. • This site interacts with the gatekeeper region to keep the pore closed. • During activation, IP3 and subsequently Ca2+ is bound and the conformational change creates a separation between the suppressor and the gatekeeper limiting interaction. • However, In the absence of IP3 this does not occur and binding of Ca2+ to the suppressor site will inhibit the opening of the channel.
IP3 and Disease Huntington’s Disease Alzheimer’s Disease
Huntington’s Disease • Genetic neurodegenerative disorder that becomes noticeable in mid-adult life • Alters muscle coordination and causes cognitive decline • Huntington’s disease affects the striatal medium spiny neurons (MSN) • Striatal MSN’s play a key role in initiating and controlling movements of the body, limbs, and eyes.
Huntington’s Disease • Huntington’s disease affects the Huntingtin (Htt) protein in the cytosol • Expanded huntingtin protein (Httexp ) has 35 additional glutamine residues in its amino terminal region • This increases the sensitivity of Type 1 IP3 receptors to IP3 • Leads to increased release of Ca2+ from the ER and causes the cytosolic and mitochondrial concentrations of Ca2+ to increase • The increased Ca2+ is thought to have caused the GABAergic MSN degeneration.
Alzheimer’s Disease • Common form of dementia which involves the progressive degeneration of the brain • The disease worsens as it progresses until it leads to death. There is currently no cure for the disease. • Early onset Alzheimer’s disease (diagnosed before 65 years of age) is linked to mutations in presenilin 1 (PS1), presenilin 2 (PS2) and amyloid precursor proteins (APP) • Mutations in PS1 show increased IP3 mediated Ca2+ release from the ER in animal models. • Disruptions in Ca2+ signaling have been shown to be a progenitor of Alzheimer’s disease
Summary Slide • IP3 releases Calcium – functions in cell proliferation, smooth muscle contraction and immune cell signalling • G-protein coupled receptors activate the PLC enzyme • PLC cleaves PIP2 into IP3 and DAG • IP3 receptors consists of 4 subunits which display positive cooperativity and allow for the release of Ca+2 ions • IP3 binds the receptor and activates it through a conformational change which reveals a Ca+2 binding site • The suppressor region also contains a Ca+2 binding site which inhibits the receptor and Ca+2 release in the absence of IP3 • Increased IP3 mediated calcium release is directly linked to Huntington’s and Alzheimer’s disease
References • SA Biosciences.Ip3 Signalling Pathway, (8, Jan., 2006) < http://www.sabiosciences.com/pathway.php?sn=IP3_Pathway> (visited 12 Oct,2012) • Taylor, C. W., Da Foncesca, P. C., & Morris, E. M. (2005). IP3 receptors: the search for structure. TRENDS in Biochemical Sciences, 29(4), 212-219. • Dawson, A. P. (1997). Calcium signalling: How do IP3 receptors work? . Dispatch, 544-547. • Hayden, M. R., & Bezprozvanny, I. (2004). Deranged neuronal calcium signaling and Huntington disease. Biochemical and Biophysical Research Communications(322), 1310-1317. • Stutzmann, G. E. (2005). Calcium Dysregulation, IP3 Signalling, and Alzheimer's Disease. Neuroscience, 110-115. • Miller T, Chamberlain P, Cooke M.(2008).Beyond IP3: roles for higher order inositol phosphates in immune cell signaling. Cell Cycle, 7(4).