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54 Cards in this Set

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G-protein coupled receptors (GPCRs)

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

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

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

GTP binding of monomeric GTPase

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

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 cova...

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.



GPCR Function

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


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

if the target of the G-protein is an enzyme, ________ molecules may be produced

second messenger

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



The synthesis and degradation of cyclic AMP

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

SEM of vibrio cholerae

different cell types respond differently to an INCREASE in cAMP

cAMP activates ____________

cyclic-AMP-dependent protein kinase (PKA)

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

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

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

PKA activation and gene transcription

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







if the target of the G-protein is an enzyme, ________ molecules may be produced

second messenger

inositol 1,4,5-triphosphate (IP 3), diacylglycerol (DAG)

target enzyme = phospholipase C- β (PLC β )



activated by G-protein G q

phospholipase C- β (PLC β )

activated by G-protein Gq

activated by G-protein Gq




phospholipase C- β (PLC β ) CLEAVES

The hydrolysis of PI(4,5)


P2 by phospholipase C-β

PIP 2 makes up HOW much of the plasma memb.

less than 1% of lipids in the plasma membrane

IP3 pathway

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

DAG pathway

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

further cleavage of DAG could be used in production of what kind of molecules?

eicosanoids (e.g. prostaglandins)

prostaglandins are responsible for what?

pain and inflammatory responses



Drugs: aspiring and ibuprofen INHIBIT prostaglandin synthesis

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

Ca2+ signalling

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)



Is GPCR involved in the release of Ca2+ by IP3 in Sperm?

NO

Ca2+ toxicity

intracellular Ca2+ = 10^-7 M (smaller)


extracellular Ca2+ = 10^-3 M (greater)



the intracellular < extracellular


How is the Ca2+ gradient maintaned

1) Na+-driven Ca2+ exchanger (plasma membrane only)
2) Ca2+ pump (uses ATP; plasma membrane & ER membrane)

1) Na+-driven Ca2+ exchanger (plasma membrane only)


2) Ca2+ pump (uses ATP; plasma membrane & ER membrane)



aht is the most commom target of Ca2+ ?

calmodulin (caM)

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



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)]

What do CaM-kinases phosphorylate?

other proteins, such as gene regulatory proteins (e.g. CREB) on serine/threonine residues

these receptors are the largest family of cell surface receptors and consist of seven transmembrane domains



which are they?

G protein-coupled receptors!

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

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

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

what if the target of the G protein is an enzyme?

then second messengers may be produced

what are examples of second messengers?

cAMP, IP3, DAG

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

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

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

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)

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)

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

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

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

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

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)

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)

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