G-Protein Signaling Introduction to intracellular signaling Dr. SARRAY Sameh, Ph.D
Cell signaling Cells communicate via extracellular signaling molecules (Hormones, growth factors and neurotransmitters ) that are produced and released by signaling cells. These molecules recognize and bind to receptors on the surface of target cells where they cause a cellular response via a signal transduction pathway Cell signaling (or signal transduction) involves: Detection of the stimulus (in most cases a molecule secreted by another cell) on the surface of the plasma membrane. Transfer of the signal to the cytoplasmic side Transmission of the signal to effector molecules. The final effect is to trigger a cell s response, such as the activation of gene transcription
Types of Signaling Three types of signaling: Depending on the distance that the signaling molecule has to travel 1. Endocrine signaling: Molecules are produce by an endocrine gland and sent through the blood stream to distant cells. 2. Paracrine signaling: The molecule affects only target cells in the proximity of the secretory cell. 3. Autocrine signaling : Cells respond to molecules they produce themselves
Types of Receptors The same ligand can bind to different receptors causing different responses (e.g., acetylcholine). On the other hand, different ligands binding to different receptors can produce the same cellular response (e.g., glucagon, epinephrine). Once a signaling molecule binds to its receptor it causes a conformational change in it. There are a number of receptor classes that are used in different signaling pathways. The two more predominant are: - G protein-coupled receptors (epinephrine, glucagon, serotonin) - Receptors with intrinsic enzymatic activity (Tyrosine Kinase Receptors)
G protein coupled receptors G-proteins are group of intracellular signaling proteins named for their ability to bind GTP; they also have GTPase activity. Two types: - heterotrimeric G proteins and - Ras super-family of G proteins (associated with RTK)
Heterotrimeric G proteins Result from the association of 3 subunits α, and (There are at least 15 forms for α subunit, each is distinguished by a subscripted letter e.g. s, i & q as Gα s, Gα i & Gα q ). GDP binds to α subunit of the inactive form when all 3 subunits are combined Signaling initiated by ligand binding to receptors called G coupled proteins receptors (GCPR).
G coupled proteins receptors: GCPR GCPR have: - Extracellular hormone binding domain, - Transmembrane region composed of 7 membrane spanning regions (helices). - Intracellular G-protein binding region
Signaling mechanism Free GCPR does not interact with G-protein close to its intracellular part. Binding of the ligand to the receptor leads to conformational change of the receptor and its binding to the G-protein complex which release GDP and bind GTP by its subunit. The G-protein now is active, and the subunit dissociate from the and subunits and the active subunit interact with an enzyme: There are 2 enzymes regulated by G proteins: adenylyl cyclase and phospholipase C.
Adenylyl cyclase 2 differents G proteins regulate the activity of adenylyl cyclase: G s stimulates its activity and G i inhibits its activity.
1- G s : -Stimulate adenylyl cyclase which uses ATP to produce camp (cyclicamp). -camp activate protein kinase A(PKA): The PKA have 2 types of subunits: regulatory (R) and catalytic (C): The - - camp binds to R subunits leading to the release of the C subunits of PKA. -The free C subunits are active and can phosphorylate PKA substrates (mostly enzymes) on their Ser and Thr residues.
2- G i : -Active G i : inhibits adenylyl cyclase and production of camp, and thus inhibit PKA activity and its substrates will not be phosphorylated.
Phospholipase C (PLC) Certain neurotransmitters, hormones and growth factors initiate signaling through G q. The After free a G hormone q activate binds phospholipase to its receptors, C, which the intracellular cleave membrane domain lipid interact phosphatidylinositol with G q 4,5bisphosphate subunit of G protein (PIP 2 ) which to release will inositol release GDP 1,4,5 triphosphate and binds GTP. (IP 3 ) in the cytosol and leave diacylglycerol The subunit (DAG) dissociates in the from membrane. the other 2 subunits, and The IP 3 binds specific receptor in the ER with release of calcium(ip 3, DAG and calcium are After the the 2 nd release messengers of the in calcium, this system). the intracellular level will increase which favors the The formation DAG of activate calmodulin-calcium a protein kinase complex C (PKC) which which is essential phosphorylate for activation cellular of proteins many that mediate enzymes. cellular responses.
Receptors with intrinsic tyrosine kinase activity Transmission of signal is via phosphorylation of tyrosine (Tyr) residues by tyrosine kinase activity which belong to the receptor itself or tyrosine kinase proteine associated with the receptor Tyr phosphatases dephosphorylate phosphotyrosines in a regulated manner to end the response to signals.
Structure of Receptors with intrinsic tyrosine kinase activity Most of catalytic receptors are single chain transmembrane proteins that associate with other single chain transmembrane proteins upon ligands binding (growth factor, cytokines and some hormones). - Each chain have 3 domains: - An extracellular ligand binding (amino or NH2 terminus) domain - -helical transmembrane region, pass through the membrane - Intracellular (cytoplasmic) region with the catalytic domain containing tyr kinase activity
2- Signaling mechanism Upon ligand binding, individual transmembrane protein come closer forming dimer: dimerization. - The dimerization leads to the activation of cytoplasmic kinase domain. - Autophosphorylation on tyrosines : As consequence, phosphorylated tyrosine residues are present on each of the receptor cytoplasmic tails. - Intracellular proteins known as adaptor proteins which contain highly conserved domains known as Src Homology 2 (SH2) and Src Homology 3 (SH3) dock with phosphotyrosines of the cytoplasmic tails of the receptor chains.
Ras super-family Ras super-family members are called small G proteins because they are monomers Ras proteins receive signals from their catalytic (enzymatic) receptors activated by ligands. Ras does not have an SH2 domain but it binds to SH2-containing adaptor molecules that bind phosphorylated residues. Ras activates a serine/threonine phosphorylation cascade called MAP kinase cascade involved in the control of growth & differentiation process.
Ras signaling mechanism Ligand binding, cause the phosphorylation of tyrosine (Tyr) residues of the receptors which act as binding site for intracellular adaptor proteins that contain SH2 domains (such as Grb2.), to form complex. SOS (Ras-specific guanine exchange factor ) join the complex, followed by Ras associated with GDP Grb2-SOS-Ras complex exchange GDP for GTP thus activating Ras. Activated Ras bind and phosphorylate Raf (a Ser protein kinase also known as MAPKKK (mitogen activation protein kinase kinase kinase). A cascade of phosphorylation reaction including several intermediate factors (MEK, MAPK..) is end with translocation of the factors into the nucleus and phosphorylation of transcription factors, leading to transcription of genes involved in early cell division.
Ras gene mutation can result in Ras protein that can not hydrolysis GTP to GDP to terminate Ras activity, leading to continuous Ras recruitment and stimulation of cell proliferation leading to malignancy
Insulin signaling Insulin signals via catalytic receptors with intrinsic Tyr kinases activity. Insulin receptor is composed of 2 extracellular subunits and 2 transmembrane subunits linked together by disulfide bridge.
Binding of insulin to its receptor results in activationof Tyr Kinase activity inducing autophosphorylation of Tyr residues in subunits These residues are recognized by phosphotyrosine binding domains (PTB) of adaptor molecules such as members of the insulin receptor substrate family (IRS). Phosphorylated IRS, activate several intracellular signaling proteins such as STATs, Ras and Pi3K. Depending on the tissue type and IRS proteins expressed, insulin will induce different biological responses to its signaling: Signal splitting
At least 4 different IRS are known: IRS-1 and IRS-2 are widely expressed IRS-3 found in adipose tissue, pancreatic cells, & liver IRS-4 found in thymus, brain & kidney
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Non-receptor Tyrosine kinases (nrtks) The second group of tyrosine kinase, are cytoplasmic enzymes: nrtks They are activated by receptors lacking Tyr Kinase activity (e.g. receptors for cytokines and some hormones like prolactin growth hormone, to carry out their signaling process nrtks regulate cell s growth, proliferation, differenciation adhesion, migration and apoptosis and are critical component in the regulation of immune system (activation of T and B cells) nrtks associate non-covalently with receptor, leading to phosphorylation of Tyr residues in the receptor tail Several nrtk have been identified but The two best caracterized are Src and Janus kinase families