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54 Cards in this Set
- Front
- Back
G-protein coupled receptors (GPCRs) |
largest family of cell-surface receptors
mediate MOST responses to signals from the extermal world and other cells
half of all known drugs work via GPCRs or their pathways
consists of seven TRANSMEMB. domains |
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General properties of Cell-Surface Receptors |
some intracellular prtns act as molecular switches
switch from an inactive to an active state, until another process switches them back to the inactive state |
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GTP binding of monomeric GTPase |
regulated by 2 proteins:
1.GTPase activating protein (GAP) mediates hydrolysis of GTP to GDP = inactivates
2.guanine nucleotide exchange factor (GEF) promotes exchange of GDP with GTP = activates |
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GPCR |
when an extracellular signal binds to a GPCR, it ACTIVATES a trimeric GTP-binding protein (G-Protein)
trimeric: α, β and γ (3 subunits)
various types of G-proteins, each specific for particular GPCRs or targets
α and γ subunits are covalently attached to lipid molec. in the plasma memb.
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GPCR Function |
1.when unstimulated α subunit has GDP bound -> inactive 2.activation of GPCR by a signal causes a conformational change that allows it to bind to the G-protein 3.activated GPCR acts like a GEF promoting the xchange of GDP w/ GTP in the α subunit-> ACTIVATING it 4.activated α and βγ subunits are released from the receptor and relay the signal to downstream targets (enzymes, ion channels)
note: ONE active receptor can activate MANY G-proteins
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How is the response (Activation of a G protein by an activated GPCR) stopped? |
α subunit is a GTPase, which hydrolyzes GTP to GDP, making it inactive
βγ subunits bind back to the inactive α subunit
receptor can also be inactivated by phosphorylation |
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if the target of the G-protein is an enzyme, ________ molecules may be produced |
second messenger |
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cAMP |
produced when the target of the G-protein is an enzyme
found in all animal cells and prokaryotes
synthesized by adenylyl cyclase (enzyme)
destroyed by cAMP phosphodiesterase (enzyme)
stimulatory G-protein (Gs) coupled to a GPCR activates adenylyl cyclase -increased cAMP production
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The synthesis and degradation of cyclic AMP |
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Cholera Toxin |
a bacterial toxin that acts on cAMP production
toxin inhibits GTPase activity of Gs
Gs would remain active adenylyl cyclase would remain active cAMP levels would remain high
prolonged cAMP in intestinal epithelial cells causes movement of Cl- and water into the gut ... leading to diarrhea |
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SEM of vibrio cholerae |
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different cell types respond differently to an INCREASE in cAMP |
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cAMP activates ____________ |
cyclic-AMP-dependent protein kinase (PKA) |
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PKA |
2 regulatory subunits + 2 catalytic subunits
when cAMP binds to the regulatory subunits, it causes the activation and release of the catalytic subunits
catalytic subunits can then phosphorylate specific target proteins on serine or threonine residues |
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if cAMP is the same in all cells that use it for signaling, and it usually acts by activating PKA, how can the same second messenger and the same PKA produce different effects in different cells? |
the substrates for PKA are different for different cell types |
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what are the effects of PKA activation? |
1. PKA can have raapid effects in seconds ex. glycogen metabolism in muscle
To turn off:
a. cAMP phosphodiesterase destroying cAMP b. protein phosphatases that dephosphorylate proteins activated by PKA
2. PKA can have slow effects in hours example: changes in gene transcription |
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PKA activation and gene transcription |
activated PKA enters the nucleus, where it activates CRE-binding prtn (CREB) by phosphorylating it
activated CREB then binds to CREB binding protein (CBP), a transcriptional coactivator ... TARGET genes are TRANSCRIBED
CREB transforms a short cAMP signal into a long-term change in the cell
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if the target of the G-protein is an enzyme, ________ molecules may be produced |
second messenger |
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inositol 1,4,5-triphosphate (IP 3), diacylglycerol (DAG) |
target enzyme = phospholipase C- β (PLC β )
activated by G-protein G q |
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phospholipase C- β (PLC β ) |
activated by G-protein Gq
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phospholipase C- β (PLC β ) CLEAVES |
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The hydrolysis of PI(4,5) P2 by phospholipase C-β |
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PIP 2 makes up HOW much of the plasma memb. |
less than 1% of lipids in the plasma membrane |
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IP3 pathway |
following cleavage by activated PLCβ, IP3 diffuses through the cytosol to the ER, where it binds to IP3 receptors called IP3-gated Ca2+-release channels
Ca2+ stored in the ER is released
rapid increase in the concentration of Ca2+ in the cytosol |
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DAG pathway |
following cleavage by activated PLCβ, DAG along with Ca2+ (released from the ER) and PtdSer (not shown) activate protein kinase C (PKC) (In the membrane)
activated PKC then phosphorylates target proteins on serine/threonine residues |
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further cleavage of DAG could be used in production of what kind of molecules? |
eicosanoids (e.g. prostaglandins) |
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prostaglandins are responsible for what? |
pain and inflammatory responses
Drugs: aspiring and ibuprofen INHIBIT prostaglandin synthesis |
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how do the DAG and IP3 signal end? |
1. IP3- can be converted to IP4 or IP2 by lipid kinases, and lipid phosphatases
2.Ca2+ can be pumped out of the cell to reduce intracellular concentrations
3. DAG can be converted to other compounds |
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Ca2+ signalling |
many signalling pathways trigger an INCR in cytosolic Ca 2+ not just in those associated w/ G-proteins
ex: Ca2+ wave during egg fertilization
Ca2+ is visualized by a fluorescent dye
wave of Ca2+ is released from the ER, sweeps across egg from the site of sperm entry
results in change in egg cell surface, prevents entry of any other sperm
sperm: sspecific form of PLC that cleaves PIP2 t IP3 and DAG
IP3 releaes Ca2+ from the ER (NO GPCR is involved)
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Is GPCR involved in the release of Ca2+ by IP3 in Sperm? |
NO |
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Ca2+ toxicity |
intracellular Ca2+ = 10^-7 M (smaller) extracellular Ca2+ = 10^-3 M (greater)
the intracellular < extracellular
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How is the Ca2+ gradient maintaned |
1) Na+-driven Ca2+ exchanger (plasma membrane only) 2) Ca2+ pump (uses ATP; plasma membrane & ER membrane)
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aht is the most commom target of Ca2+ ? |
calmodulin (caM) |
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Calmodulin(caM) |
- most common target of Ca2+ 4 binding sites for calcium
when activated by at least 2 or more ca2+ binding, it undergoes a conformational change that allows it to bind to active target proteins
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Target proteins of Calmodulin |
Target Proteins:
1. enzymes 2. membrane-bound transport proteins (e.g. Ca2+ pumps) 3. protein kinases [e.g. Ca2+/calmodulin-dependent kinases (CaM-kinases)] |
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What do CaM-kinases phosphorylate? |
other proteins, such as gene regulatory proteins (e.g. CREB) on serine/threonine residues |
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these receptors are the largest family of cell surface receptors and consist of seven transmembrane domains
which are they? |
G protein-coupled receptors! |
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G protein shape and structure |
trimeric: alpha, beta, and gamma subunits
alpha and gamma subunits are covalently attached to lipid molecules in the plasma membrane |
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GPCR pathway |
1) when unstimulated, alpha subunit has GDP bound (inactive)
2) activation of GPCR by a signal causes a conformational change that allows it to bind to the G protein
3) activated GPCR then acts like a GEF, promoting the exchange of GDP with GTP in the alpha subunit, activating it
4) activated alpha and beta-gamma subunits are released from the receptor and relay the signal to downstream targets (e.g. enzymes, ion channels)
N.B. one active receptor can activate many G proteins |
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how is the GPCR response stopped? |
alpha subunit is a GTPase, which hydrolyzes GTP to GDP, making it inactive
bet-gamma subunits bind back to the inactive alpha subunit
receptor can also be inactivated by phosphorylation |
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what if the target of the G protein is an enzyme? |
then second messengers may be produced |
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what are examples of second messengers? |
cAMP, IP3, DAG |
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cAMP background info (incl. how it's synthesized & how it's destroyed); disease associated with it |
cyclic AMP (cAMP) -found in all animal cells and prokaryotes -synthesized by adenylyl cyclase -destroyed by cAMP phosphodiesterase -stimulatory G protein (Gs) coupled to a GPCR activates adenylyl cyclase (increases cAMP production)
cholera toxin -inhibits GTPase activity of Gs -keeps Gs, adenylyl cyclase active; cAMP levels remain high -prolonged cAMP in intestinal epithelial cells causes movement of chloride and water into the gut, leading to diarrhea
DIFFERENT CELL TYPES RESPOND DIFFERENTLY TO AN INCREASE IN cAMP |
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what does cAMP activate? |
cAMP-dependent protein kinase (PKA)
PKA = 2 regulatory subunits + 2 catalytic subunits
when cAMP binds to the regulatory subunits, it causes the activation and release of the catalytic subunits -catalytic subunits can then phosphorylate specific target proteins on serine or threonine residues |
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if cAMP is the same in all cells that use it for signalling, and if it usually acts by activating PKA, how can the same second messenger and the same PKA produce different effects in different cells? |
the substrates for PKA are different for different cell types |
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what are the effects of PKA activation? |
1) PKA can have rapid effects in seconds (e.g. glycogen metabolism in muscle) -can be turned off by a) cAMP phosphodiesterase destroying cAMP b) protein phosphatases that dephosphorylate proteins activated by PKA
2) PKA can have slow effects in hour (e.g. changes in gene transcription) |
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effects of PKA activation on gene transcription |
-activated PKA enters the nucleus, where it activates CRE-binding protein (CREB) by phosphorylating it -activated CREB then binds to CREB-binding protein (CBP), a transcriptional coactivator -both CREB and CBP bind DNA at a region near genes to be transcribed called a cAMP response element (CRE) |
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recall that if target of G protein is an enzyme, second messengers may be IP3 and DAG
what's the target enzyme? IP3 and DAG activated by what?
what does the target enzyme do? |
target enzyme: phospholipase C-beta (PLC-beta)
activated by G protein Gq
target enzyme PLC-beta cleaves PIP2 into IP3 (hydrophilic; soluble in cytosol) and DAG (hydrophobic; stays in membrane)
N.B. PIP2 makes up less than 1% of lipids in the plasma membrane |
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IP3 pathway |
following cleavage by activated PLC-beta, IP3 diffuses through the cytosol to the ER, where it binds to IP3 receptors called IP3-gated Ca2+-release channels -Ca2+ stored in the ER is released -rapid increase in concentration of Ca2+ in cytosol |
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DAG pathway |
following cleavage by activated PLC-beta, DAG along with Ca2+ (released from ER) and PtdSer activate protein kinase C (PKC)
-activated PKC phosphorylates target proteins on serine/threonine residues
DAG can be further cleaved and used in the production of signalling molceules called eicosanoids (e.g. prostaglandins) -prostaglandins are responsible for pain and inflammatory responses -drugs like Aspirin and ibuprofen inhibit prostaglandin synthesis |
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how do the signals end for IP3 and DAG pathways? |
1) IP3 can be converted to IP4 or IP2 by lipid kinases and lipid phosphatases, respectively
2) Ca2+ can be pumped out of the cell to reduce intracellular concentrations
3) DAG can be converted to other compounds |
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what's an example of a pathway that triggers an increase in cytosolic Ca2+ other than a pathway associated with G proteins? |
Ca2+ wave during egg fertilization -wave of Ca2+, released from the ER, sweeps across the egg from the site of sperm entry -results in a change in the egg cell surface that prevents the entry of other sperm -sperm has a specific form of PLC that cleaves PIP2 to IP3 and DAG -IP3 releases Ca2+ from the ER (no GPCR is involved) |
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Ca2+ is toxic to cells. how is the Ca2+ gradient maintained? |
1) Na+-driven Ca2+ exchanger (plasma membrane only)
2) Ca2+ pump (uses ATP; plasma & ER membranes) |
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what is the most common target of Ca2+?
how many Ca2+ binding sites does it have?
how does it activate target proteins?
what do target proteins include? |
calmodulin (CaM)
four binding sites for Ca2+
when activated by at least two Ca2+, CaM undergoes a conformational change that allows it to bind to and activate target proteins
target proteins include a) enzymes b) membrane-bound transport proteins (e.g. Ca2+ pumps) c) protein kinases (e.g. Ca2+/calmodulin-dependent kinases (CaM-kinases)) -CaM-kinases phosphorylate other proteins such as regulatory proteins (e.g. CREB) on serine/threonine residues |