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86 Cards in this Set
- Front
- Back
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Important/common second messengers
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cAMP, cGMP, Ca2+, inositol 1,4,5-triphosphate (IP3), diacylglycerol (DAG)
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ultimate effect of the signal pathway
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activate/inhibit pumps, channels, enzymes, transcription factors that directly control metabolic pathways, gene expression, membrane permeability
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Principes of signal transduction
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1. release of the primary messenger
2. reception of the primary messenger 3. delivery of the message inside the cell by the second messenger 4. activation of effectors that alter the physiological response 5. termination of the signal |
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epinephrine
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-hormone secreted by the adrenal glands
-exerts 'fight or flight' response -begins with ligand binding to a B-adrenergic receptor (B-AR). (member of 7TM receptors - largest class of cell-surface receptors) |
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Activation of PKA by G-protein pathway
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1. epinephrine binds to B-AR
2. conformational change in B-AR activates a G protein 3. activated G protein stimulates the activity of adenylate cyclase 4. cAMP carries signal in the cell |
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What type of receptor is rhodopsin?
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7TM
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rhodopsin
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A single lysine residue is covalently modified by a form of vitamin A, 11-cis-retinal, near the extracellular side of the receptor. Exposure to light induces the isomerization of 11-cis-retinal to its trans form, producing a structural change in the receptor
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What is rhodopsin similar to?
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B-adrenergic receptors
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The conformational changes in the 7TM receptor activates a protein called
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a G protein (it binds guanyl nucleotides)
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The activated G protein stimulates the activity of - , an enzyme that catalyses the conversion of - into -.
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adenylate cyclase catalyses the conversion of ATP into cAMP
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In its unactivated state, the G protein is bound to -. In this form, the G protein exists as a -mer consisting of -,-,and- subunits.
The - subunit binds the nucleotide. This subunit is a member of the - family, and the - participates in nucleotide binding. The - and - subunits are usually anchored to the membrane by covalently attached fatty acids. |
GDP
heterotrimer a, B, y a subunit (Ga); P-loop NTPase family; P-loop a and y subunits usually anchored to the membrane |
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THE ROLE OF THE HORMONE-BOUND (7TM) RECEPTOR IS TO
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CATALYZE THE EXCHANGE OF GTP FOR BOUND GDP, SO THAT GTP IN THE CELL CAN DISPLACE GDP IN THE NUCLEOTIDE-BINDING SITE
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On GTP binding, the a subunit simultaneously dissociates from the - dimer (-), transmitting the signal.
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By dimer (GBy)
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A SINGLE HORMONE-RECEPTOR COMPLEX CAN STIMULATE - - IN MANY - PROTEIN -MERS.
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NUCLEOTIDE EXCHANGE; G-PROTEIN; HETEROTRIMERS
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WHICH SUBUNIT IS A MEMBER OF THE P-LOOP NTPASE FAMILY?
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A SUBUNIT
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-'s of Ga molecules are converted from their GDP form into their GTP form for each bound molecule of hormone, giving an - response.
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hundreds; amplified
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7TM receptors are often called this because they signal through G-proteins
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G-protein-coupled receptors (GPCRs)
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Once Ga has changed its conformation from the GDP form, it longer has a high -------. This surface is now exposed for binding to other proteins.
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affinity for Gby
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In the B-AR pathway, the new binding partner is -.
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adenylate cyclase, the enzyme that converts ATP into cAMP
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adenylate cyclase
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is a MEMBRANE protein that contains 12 membrane-spanning helices; two large CYTOPLASMIC DOMAINS form the catalytic part of the enzyme
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The interaction of - with adenylate cyclase favors a more catalytically active conformation of the enzyme, stimulating - production.
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Ga; cAMP production
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The Ga subunit that participates in the - pathway is called -.
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B-AR; Gas (s for stimulatory)
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The binding of epinephrine to the receptor on the cell surface increases the rate of - production - the cell.
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cAMP; inside
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Why can cAMP influence so many cellular process?
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They activate protein kinase A (PKA)
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PKA consists of -.
In the absence of cAMP, the - complex is catalytically -. |
2 regulatory (R) chains and 2 catalytic (C) chains;
R2C2; inactive |
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Activated PKA then phosphorylates specific - and - residues.
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Ser and Thr
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PKA stimulates the expression of specific genes by
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phosphorylating a transcriptional activator called the cAMP response element binding (CREB) protein.
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The bound GTP in Ga acts as a
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clock that spontaneously resets the Ga subunit after short time period
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After GTP hydrolysis and the release of Pi, the - bound form of Ga then reassociates with - to reform the inactive heterotrimeric protein.
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GDP; GBy
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How is B-AR reset?
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1. The hormone dissociates, returning the receptor to its initial unactive state.
2. The signaling cascade activates a KINASE that phosphorylates serine and threonine residues in the carboxyl-terminal tail of the receptor. This results in deactivation of the receptor |
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B-adrenergic-receptor kinase (G-protein receptor kinase 2, GRK2)
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phosphorylates the carboxyl-terminal tail of the hormone-receptor complex
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B-arrestin
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binds to the phosphorylated receptor and further diminishes its ability to activate G proteins
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phosphoinositide cascade
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converts extracellular signals into intracellular ones - the intracellular messengers formed by activation of this pathway arise from the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2)
-angiotension II hormone that controls blood pressure |
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B-AR activates G protein -.
angiotension II receptor activates -. |
Gas; Gaq
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phospholipase C
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Gaq in its GTP form binds and activates the B isoform of this enzyme. This enzyme catalyses the cleavage of PIP2 into the 2 second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)
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IP3
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-is soluble in diffuses away from the membrane
-causes the rapid release of Ca2+ from the intracellular stores in the ER (Ca2+ reservoir by Ca ATPase) -IP3-gated Ca2+ channels in the ER open to allow Ca2+ to flow from the ER into the cytoplasm |
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Once Ca2+ flows from the ER into the cytoplasm
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-it can bind proteins including calmodulin and PKC
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elevated cytoplasmic Ca2+ levels triggers processes such as
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smooth-muscle contraction, glycogen breakdown, and vesicle release
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DAG remains in the - and activates -.
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plasma membrane; PKC
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protein kinase C (PKC)
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phosphorylates Ser and Thr in many target proteins
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How does PKC bind DAG?
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-PKC requires bound Ca2+
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How do IP3 and DAG work in tandem?
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IP3 increases [Ca2+] and Ca2+ facilitates the DAG-mediated activation of PKC
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DAG
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diacylglycerol
activates PKC |
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Why is Ca2+ a widely used second messenger?
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1. small changes in Ca2+ readily detected
-Ca2+ conc. kept low because it can form poorly soluble salts 2. it can bind tightly to proteins and induce structural rearrangement - glu, asp residues, carbonyl, gln, asn -it can be coordinated to multiple ligands (6 to 8 oxygen atoms) to cross-link segments of protein and induce conformational change |
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The capacity of Ca2+ to be coordinated to multiple ligands
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enables it to cross-link different segments of a protein and induce sig. conformational changes
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Fura-2
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binds Ca2+ through oxygen atoms and changes fluorescent properties upon binding
-molecular-imaging agents |
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calmodulin (CaM)
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EF-hand protein with 4 Ca2+ binding sites
-Ca2+ sensor -activated at ~500 nM Ca2+ conc. |
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EF hand
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helix, loop, second helix
-7 O atoms coordinated to Ca2+ |
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How does Ca2+-calmodulin complex stimulate target proteins?
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induces structural rearrangements in these binding partners
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calmodulin-dependent protein kinases (CaM kinases)
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phosphorylate many different proteins and regulate fuel metabolism, ionic permeability, neurotransmitter synthesis and release
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The recurring theme in signal-transduction pathways:
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1. second messenger conc. is increased
2. signal is sensed by second-messenger binding protein 3. second messenger binding protein acts to generate changes in enzymes. |
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insulin
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peptide hormone released in response to increased blood-glucose levels
-2 chains, linked by 3 disulfide bonds |
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insulin receptor
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dimer of 2 identical units - 1 a chain and 1 B chain
the two z subunits move together to form a binding site for a single insulin molecule |
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What type of kinase is the kinase domain in the insulin receptor dimer subunits? How is it different from PKA?
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tyrosine kinase - PHOSPHORYLATES TYR, NOT SER OR THR
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insulin receptor is also referred to as - because the tyrosine kinase is a component of the receptor itself
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receptor tyrosine kinase
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As insulin binds, the 2 -- are drawn together. As they are forced together, the kinase domains catalyse the addition of - P groups from - to -.
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B subunits; 2; ATP; Tyr
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Additional phosphorylation at other sites of the insulin receptor act as
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docking sites for other substrates - insulin-receptor substrates
-IRS-1 and IRS-2 |
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pleckstrin homology domain
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amino-terminal part of IRS-1 and 2 which binds phosphoinositide
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What domains act together to anchor the IRS to the insulin receptor?
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pleckstrin homology domain
and phosphotyrosine-binding domain |
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Each IRS protein contains 4 sequences of the form -. When the - residues in the sequences are phosphorylated, IRS molecules can act as - proteins.
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Tyr-X-X-Met
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Phosphotyrosine residues are recognized by
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Src homology 2 (SH2) domains in proteins
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Which proteins containing SH2 domains recognize IRS proteins?
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phosphoinositide 3-kinases (PI3Ks)
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Mechanism of insulin signaling from IRS binding
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1. PI3K binds to IRS and is drawn to the membrane
2. adds a phosphoryl group to the 3 position of inositol in PIP2 to form PIP3 in the membrane 3. PIP3 activates protein kinase PDK1 by a pleckstrin domain 4. PDK1 moves into the cell and phosphorylates Akt that phosphorylates targets that control trafficking of the glucose receptor GLUT4 |
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How is EGF receptor similar to insulin receptor? How is it different?
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It is a dimer of 2 identical subunits. However, the subunits exist as monomers until they each bind EGF.
It also undergoes cross-phosphorylation by another unit within the dimer. Unlike the insulin receptor, the site os phosphorylation is not in the active site bu in the C-terminal side, where up to Tyr are phosphorylated |
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dimerization arm
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where each monomer reaches out and inserts into a binding pocket on the other monomer
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Why doesn't the receptor dimerize and signal in the absence of EGF?
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The dimerization arm binds to a domain within the same monomer in its closed configuration and is poised to bind ligand
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Her2
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a receptor similar to EGF receptor that adopts a structure similar to ligand-bound EGF. It forms heterodimers with the EGF receptor and participates in cross-phosphorylation reactions, leading to overexpression by forming homodimers that signal without ligand.
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How EGF activates Ras
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1. SH2 domains on proteins dock on phosphotyrosines on EGF receptors. Grb-2's SH2 binds to a phosophotyrosine residue
2. Grb-2's 2 SH3 domains bind Sos 3. Sos binds Ras and activates it The binding of EGF to its receptor leads to the conversion of Ras into its GTP form through Grb-2 and Sos intermediates |
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Ras
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member of the small G proteins class.
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How is Ras similar to G proteins? How are they different? What is their relationship?
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The small G protein contains bound GDP in unactivated forms. Sos opens up the nucleotide-binding pocket of Ras, allowing GDP to be replaced by GTP.
Small G proteins are monomeric, whereas G proteins are heterotrimeric. They are related by divergent evolution. |
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What is Sos also known as?
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guanine-nucleotide-exchange factor (GEF)
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Ras activated a protein kinase cascade
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In GTP form, Ras binds other proteins, including PK Raf in the membrane. Activated Raf phosphorylates MEKs (kinases). MEKs activate activate kinases called extracellular signal-regulated kinases (ERKs). ERKs phosphorylate transcription factors in the nucleus and other PK's
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Signal amplification by PK's
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They often phosphorylate multiple substrates
-epinephrine: cAMP-dependent PKA turns increase of cAMP into covalent modification of enzymes -insulin/EGF: receptors are PK's and more PK's are downstream |
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second messengers
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cAMP, Ca2+, IP3, DAG
-activated by enzymes or ion channels |
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specialized domains
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pleckstrin homology domains: facilitate protein interactions with lipid PIP3
SH2: mediate interactions with polypeptides containing phosphorylated TYR SH3: interact with PRO-containing sequences |
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Evolution of signal transduction pathways
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They evolved largely by incorporation of DNA fragments encoding specialised domains into genes encoding pathway components
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Causes of cancer
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-mutation of kinases - no regulation
-mutation of RAS - no GTPase -mutation of phosphatases - no tumor suppression -EFGR mutation doesn't require ligand to activate -(HER2)2 always on |
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Whooping cough
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-toxin ADP ribosylates Gai protein
-GTP hydrolysis very fast (always off) -can't inhibit adenylate cyclase |
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Cholera
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-toxin GDP ribosylates Gas protein
-stabilizes GTP (always on) PKA too active, Cl- channel open, Na/H pump off -loss of NaCl and water |
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overview of signal transduction
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outside->inside->physiological effect->turn off->reset
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Why should chemists care about signal transduction?
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1. potentially many PHARMA
2. interesting protein/protein int., regulation and evolution |
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v-src
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oncogene
-leads to the generation of cancerlike characteristics -carried by Rous sarcoma virus |
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v-Src
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protein encoded by v-src gene
-protein tyrosine kinase with SH2 and SH3 domains |
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c-src
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pro-oncogene
-when mutated, can be converted into an oncogene |
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Why is v-src always active and c-src not?
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The C-terminus lacks the critical tyrosine residue
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monoclonal antibodies
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target EGFR receptors
-compete with EGF for binding site, blocks change in conformation that exposes dimerization arm -EGFR-controlled pathway is not initiated |
