21G Protein Signal Cascade A hormone (e.g., epinephrine or glucagon), that activates formation of cyclic AMP, binds at the cell surface to a receptor with seven transmembrane a-helices. Rhodopsin, depicted at right, was the first member of the family of 7-helix receptors to have its structure determined by x-ray crystallography.A 7-helix receptor interacts with a G-protein, a heterotrimeric GTP-binding protein. A G-protein has 3 subunits, designated a, b, g. The 7-helix receptors that interact with G-proteins are called GPCR, or G-Protein-Coupled Receptors. Various proteins interact with GPCRs to modulate their activity. Effects of these interactions include altered ligand affinity, receptor dimerization that may enhance or alter activity, altered receptor localization, etc. If part of a pathway that stimulates Adenylyl Cyclase, a G-protein is called Gs, and its a subunit Gsa22Rhodopsin was the first member of the family of 7-helix receptors to have its structure determined by X-ray crystallography. Rhodopsin PDB 1F88 G Protein Signal CascadeA hormone (e.g., epinephrine or glucagon) that activates formation of cAMP, binds at the cell surface to a receptor with 7 transmembrane α-helices.23 Rhodopsin PDB 1F88 A 7-helix receptor interacts with a G-protein, a heterotrimeric GTP-binding protein. The 3 subunits of a G-proteinare designated α, β, γ. The 7-helix receptors that interact with G-proteins are called GPCR, or G-Protein-Coupled Receptors.24A G-protein’s α subunit (Gα) binds GTP, & can hydrolyze it to GDP + Pi. α & γ subunits have covalently attached lipid anchors that bind a G-protein to the plasma membrane cytosolic surface.Adenylate Cyclase (AC) is a transmembrane protein, with cytosolic domains forming the catalytic site.AC hormone signal outside GPCR plasma membrane GTP GDP ATP cAMP + PPi α γ γ + α cytosol GDP β β GTP A G protein that is part of a pathway that stimulatesAdenylyl Cyclase, is called Gs& its α subunit Gsα.25The sequence of events by which a hormone activates cAMP signaling:1. Initially Gαhas bound GDP, and α, β, & γsubunits are complexed together. AC hormone signal outside GPCR plasma membrane GTP GDP ATP cAMP + PPi α γ γ + α cytosol GDP β β GTP The complex of β & γsubunits Gβ,γinhibits Gα.262. Hormone binding to a 7-helix receptor (GPCR) causes a conformational change that is transmitted to the G protein. The nucleotide-binding site on Gαbecomes more accessible to the cytosol, where [GTP] > [GDP].Gαreleases GDP & binds GTP (GDP-GTP exchange). AC hormone signal outside GPCR plasma membrane GTP GDP ATP cAMP + PPi α γ γ + α cytosol GDP β β GTP273. Substitution of GTP for GDP causes another conformational change in Gα. Gα-GTP dissociates from the inhibitory βγ complex & can now bind to and activate Adenylyl Cyclase.AC hormone signal outside GPCR plasma membrane GTP GDP ATP cAMP + PPi α γ γ + α cytosol GDP β β GTP284. Adenylyl Cyclase, activated by Gα-GTP, catalyzes synthesis of cAMP.5. Protein Kinase A (cAMP Dependent Protein Kinase) catalyzes phosphorylation of various cellular proteins, altering their activity.AC hormone signal outside GPCR plasma membrane GTP GDP ATP cAMP + PPi α γ γ + α cytosol GDP β β GTP29Turn off of the signal:1. Gαhydrolyzes GTP to GDP + Pi. (GTPase).The presence of GDP on Gαcauses it to rebind to the inhibitory βγ complex. Adenylate Cyclase is no longer activated.2. Phosphodiesterase catalyzes hydrolysis of cAMP Æ AMP.3. Hormone receptor desensitization occurs. This process varies with the hormone and will not be discussed here.4. Protein Phosphatase catalyzes removal by hydrolysis of phosphates that were attached to proteins via Protein Kinase A.30Insulin activates the G-protein Ras via the Insulin Receptor.Ras activate Raf and Rafactivates a MAP kinase cascade.The MAP kinase cascade activates transcriptional activators and genes are “turned on”31Signal amplification is an important feature of signal cascades:  One hormone molecule can lead to formation of many cAMP molecules. Each catalytic subunit of Protein Kinase A catalyzes phosphorylation of many proteins during the life-time of the cAMP.32The stimulatory Gsα, when it binds GTP, activates Adenylyl cyclase.An inhibitory Giα, when it binds GTP, inhibitsAdenylyl cyclase. Different effectors & their receptors induce Giαto exchange GDP for GTP than those that activate Gsα. In some cells, the complex of Gβ,γthat is released when Gαbinds GTP is itself an effector that binds to and activates other proteins.33Signal transduction cascades Lead to signal amplificationSignal amplification is an important feature of signal cascades.One hormone molecule can lead to formation of many cAMP molecules.Each catalytic subunit of Protein Kinase A catalyzes phosphorylation of many proteins during the life-time of the cAMP. of G-Protein Signal CascadeThe stimulatory Gsa, when it binds GTP, activates Adenylyl Cyclase. An inhibitory Gia, when it binds GTP, inhibits Adenylyl Cyclase. Different effectors and their receptors induce Gia to exchange GDP for GTP than those that activate Gsa (diagram p. 677). In some cells, the complex of Gb,g that is released when Ga binds GTP is itself an effector that binds to and activates other proteins.34Glucagon (liver)Epinephrine (muscle)Phosphorylase bRequires AMP“Less active form”Difference?Phosphorylation3 enzymes involvedPhosphorylase aDoes not require AMPFor activity“Active form”Glycogen breakdownPhosphorylase kinase-phosphorylates phosphorylase b at ser-14 to MAKE phosphorylase acAMP dep protein kinase phosphorylates the kinase and activates itPhosphoprotein pyrophospatasedephosphorylates phosphorylase aand the