BIOL 2020 Lecture Notes - Lecture 8: G Protein–Coupled Receptor, Protein Kinase A, Adenylyl Cyclase
Cell Signaling and Signal Transduction: Communication Between/Within Cells
November 2-16, 2015
Read: pages 617-663
• Signaling regulates important processes within the cell
• Ex. Growth/division, differentiation, metabolism
• Cancer and other diseases can be resulted from mutations that prevent proper signalling
response
Signaling Pathways – General Features:
• Extracellular signaling molecule remain outside the cell even as they interact with the inside
o May be referred to a ligand
o Include information with a specific function
• The signaling molecule binds to a transmembrane receptor and causes a conformational change
in the protein which is transmitted to the cytoplasmic side of membrane
• The protein can now activate other proteins within the cell
• ONE PATHWAY:
o Turns on effector molecule (enzyme)
o Enzyme produces second messengers
o Second messengers go on to produce changes within the cell
• SECOND PATHWAY:
o Changes conformation so other proteins can bind on and become activated so they can
go on to activate other molecules with the cell in a series
o
• Information outside the cell is passed along a series to proteins (signal pathway) to activated
target protein to affect some process in the cell
• Functions:
o Transcription
o Survival
o Protein synthesis
o Movement
o Cell death
o Metabolic change
• Eventually the protein is deactivated and terminated
• Each time a protein is activated/deactivated within the series they undergo a conformational
change
• Most proteins within the pathway are either kinases (phosphorylation) or phosphatases (cuts
off phosphate group)
G Protein-Coupled Receptors: Second Messengers
• Large group of receptors in the cytoplasmic membrane (integral proteins) that bind ligand
• Coupled to a gene protein – produce second messengers
• One signal protein – crosses the membrane 7 times (in alpha-helix form)
o 3 loops on extracellular side (attached to NH2)
▪ corresponds to ligand
o 3 loops on cytoplasmic side (attached to COOH)
▪ form a binding side for G proteins
• When information passes across membrane, the loops change conformation and increase its
affinity with the G protein causing it to bind
• G protein: heterotrimers
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o Alpha, beta and gamma subunits
o Alpha and gamma insert lipids into the membrane for attachment
o Protein can bind to GTP (activation) or GDP (deactivation)
• G protein interacts with effector protein (adenylyl cyclase) to produce intracellular second
messengers (cyclic AMP)
Receptor-mediated activation of effectors by heterotrimeric G proteins:
• Receptor on membrane is not activated; G protein is bound to GDP
• Ligand binds to receptor and causes conformational change that is transferred across the
membrane
• The change is conformation increases the receptor’s affinity for the G protein
• The G protein binds to the receptor and GTP replaces GDP on the alpha subunit
• *One activated receptor can turn on various gene proteins = signal amplification
• When GTP binds the G protein undergoes a conformational change and dissociates from the
receptor
• The alpha subunit with GTP bound dissociates from the other subunits of the G protein
• Alpha binds to effector (ex. Adenylyl cyclase) which produces second messenger (cAMP)
• *Signal amplification
• Alpha subunit of G protein cuts phosphate off of GTP to produce GDP+P
• Regulators of G signalling protein binds on to alpha subunit to speed up the GTP hydrolysis
• Forms a heterotrimer as its affinity increases
To turn off receptor:
• G-protein coupled receptor kinase phosphorylates receptor (ATP-ADP on protein) so phosphate
groups are attached to the receptor
• When receptor is active it an attach to kinase easily but is less able when phosphorylated
• Another protein, arrestin, binds to the phosphates on the receptor so it can no longer stimulate
the cell = desensitization
• Receptor can be internalized or destroyed * Clathrin – AP2 Endocytosis
Adenylyl Cyclase:
• Enzyme/effector/integral membrane protein that produces cAMP
• Formed by 6 alpha helicase and another 6 that come together to form an active site(attached to
COOH)
• The active site can bind ATP (cuts off 2 phosphates from ATP and cyclizes what is left to create
cAMP)
Protein Kinase A (PKA) Activation
• Production of cAMP activates kinase
• Inactive PKA: heterotetromore = nucleotide binding site + catalytic side
• cAMP binds to regulators which dissociate from catalytic sites
• Catalytic sites may be used in a signalling pathway
• *Undergoes allosteric change
Phospholipid-Derived Second Messengers:
• originate from phospholipids
• phospholipases – lipid splitting enzymes **
• phospholipid kinases – lipid phosphorylating enzymes
• phospholipid phosphatases – lipid dephosphorylating enzyme (cleave phosphate groups off)
• These enzymes are involved in the production of secondary messengers by modifying
phospholipids
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**Phospholipases:
• Ester bonds + diacylgylercol
• Phospholipase C – cut ester bonds that create the phospholipids
o Splits into 2 parts: charged head group + Diacylglycerol
Phosphatidylinositol (PI) Derived Second Messengers:
• Second messengers are generated when lipids get cleaved
• Fatty acid chain is linked to PI on membrane through head group
• Kinase adds additional phosphates to a the inositol ring
• Gives a variety of phosphorylated PI = phosphoinositides
• Phosphorylation occurs on ring in cytosol, not within the membrane
• Kinase can ass more phosphates to create phosphatidylinositol 4,5-biphosphate (PIP2)
• Ex. PIP2 and phosphorylated derivatives is recognized by a PH domain and bind close to
membrane
o This activates the PH protein
o A G protein coupled receptor responds to a ligand outside the cell and causes the
attachment of enzymes with PH domain to attach to phosphates (phosphatidylinositol-
specific phospholipase C- **PI-PLC)
o Protein can be activated to have an association near the membrane
o Enzyme hydrolyzes PIP2 to cut it into inositol-1,4,5-triphosphate (IP3) and
diacylglycerol (DAG) *from one second messenger 2 second messengers
=accumulation
o Diacylglycerol – 2 fatty acids with glycerol backbone
▪ Remains within the membrane and binds to activate protein kinase C
▪ Protein kinase C goes on to phosphorylate other proteins within the cell
o IP3 – sugar phosphate; soluble with in the cytosol
▪ Diffuses through the cytosol and binds to the IP3 receptor on the smooth ER
▪ Receptor undergoes conformational change
▪ The receptor is a membrane channel for the movement of Ca++ into the cytosol
▪ Calcium is another second messenger
o Calcium – pages 648-653
▪ Ca++ channels – control concentration of calcium in the cytosol
▪ The concentration of calcium in the cytosol is significantly less than
concentrations outside the cell or in the smooth ER
▪ Channels work to keep concentration low
▪ ATP-driven Ca++ transport system
▪ Acts as second messenger when its within the cytosol
▪ Not always a messenger:
• Has to bind to calmodulin (binds for 4 calciums)
• When one calcium binds, the affinity for calcium at the next site
increases
• The affinity of calcium binding proteins is relatively low at low
concentrations within the cytosol
• If the concentration of calcium within the cytosol is increased, there is a
larger chance to bind to calmodulin to activate the calcium binding
protein
• Activities:
o Affects protein kinase – activated by calmodulin
o Calmodulin can activate cyclic nucleotide phosphodiesterase to
influence the amount of cyclic Amp within the cell
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Document Summary
Cell signaling and signal transduction: communication between/within cells. Read: pages 617-663: signaling regulates important processes within the cell, ex. Growth/division, differentiation, metabolism: cancer and other diseases can be resulted from mutations that prevent proper signalling response. Signaling pathways general features: may be referred to a ligand. *one activated receptor can turn on various gene proteins = signal amplification: when gtp binds the g protein undergoes a conformational change and dissociates from the receptor. *signal amplification: the alpha subunit with gtp bound dissociates from the other subunits of the g protein, alpha binds to effector (ex. Adenylyl cyclase: enzyme/effector/integral membrane protein that produces camp, formed by 6 alpha helicase and another 6 that come together to form an active site(attached to. Cooh: the active site can bind atp (cuts off 2 phosphates from atp and cyclizes what is left to create camp)
