Intracellular Receptors Extracellular Receptor 8 Once a ligand binds to a receptor stuff happens... Ligand Y G A Receptor Cai X Z 9 Lipophobic Signals
Extracellular signals are transduced and amplified
Process by which a signal crosses the membrane
Process by which the effect of a signal is multiplied
Occurs one of two ways
via direct phosphorylation
Ligand (eg a hormone) Protein Receptor Protein Phosphate Group An example we will discussRTK Receptors Or... 11 Transduction
A far more common form of transduction is via G proteins ...
A G Amplifying Enzyme G protein Ligand Receptor We will focus on these types of mechanisms 12 Amplifyng Enzymes
cAMP cGMP Phospholipase C. IP3
Activate second messenger cascades
Amplifying Enzyme 1st Order Second Messenger 2nd Order Second Messenger 3rd Order Second Messenger 13 So What
Steps in the cascade allow amplification of the signal
A few hormone molecules activate numerous enzymes.
e.g. each PKA molecule (a second messenger) can activate several phosphorylase kinase molecules.
PKA has three different and coordinated effects. Effects - key to coordination of separate responses by the cell
14 Focus on G Protein-Mediated Signaling 15 G-Protein Coupled Receptors
Many hormones work through GPCRs
As many as 2000 different GPRCs
many not yet known genes identified
no known ligand yet
1/20 of all the proteins are GPCRs
one quarter of all prescription drugs work through GPCRs
16 G-Protein Coupled Receptors
A family of integral proteins all have seven trans-membrane a-helix segments
All work in the same way
through the Heterotrimeric G-proteins
turn on effector molecule which makes the second messenger
epinephrine and glucagon (e.g.) turn on adenylyl cyclase to make cAMP 2nd messg.
others (e.g. acetylcholine) use phosphoinositol DAG second messengers
others (e.g. photoreceptors) use cyclic GMP
G protein action
1. When receptor combines with ligand receptor changes shape and binds the a subunit of the G protein
2. Activation of the receptor a subunit then exchanges a GDP for a GTP entering activated state
- the receptor/ligand can activate several G proteins as long as ligand is bound
3. Relay the a subunit dissociates from bg and associates with effector producing second message
- bg stay together
- second message is made for duration of binding
18 G Protein Action receptor ligand g a b 1. GDP Heterotrimeric G-protein GTP 2. GDP exchange 19 G protein action g a Effector. (such as adenylyl cyclase or PI-PLCb) b GTP 3. Second Message 20 G-proteins Have Different Effects
Four types of G-proteins different Ga subunits.
Gas stimulates adenylyl cyclase
Gaq activates PLCb
Gai inactivates adenylyl cyclase
Ga12/13 activates Src Ras phospholipase D and protein kinase C
The Gbg complex can also activate other effectors.
21 Integrated Example
Glucagon and Epinephrine Signaling
22 A word from our sponsors glucagon and epinephrine effects on glucose
Utilization of glucose
primary energy source
stored as insoluble polymer glycogen
glycogen to glucose is promoted by hormones
glucagon (released from pancreas) boosts blood glucose
epinephrine (adrenal gland) boosts blood glucose during stress
Promote breakdown of glycogen to glucose-1-phosphate first step in catabolism
Glucose is either catabolized or sent to bloodstream for delivery to other places.
Inhibit glycogen synthase this enzyme makes glycogen so it has to be turned off in order for the cells to release or burn glucose.
23 The Process
1. Hormone binds to receptor and heterotrimeric G-protein
2. Activation of effector Adenylyl cyclase formation of cAMP diffuses into cytoplasm
3. cAMP binds to allosteric site activating Protein kinase A (PKA)
4. This phosphorylates target Glycogen synthase inactivating it. Glycogen no longer produced
5. At same time phosphorylates enzyme Phosphorylase kinase activating it
24 The Process contd
6. the phosphorylase kinase then phosphorylates target enzyme Phosphorylase activating it
7. phosphorylase catalyzes glycogen break-down glucose-1-phosphate is released
8. also at same time in the nucleus PKA phosphorylates transcription factor cyclic AMP response element binding protein (CREB)
9. phosphorylated CREB dimerizes and binds to cAMP response element (CRE) turning on PEPCK gene gluconeogenisis increases
25 Glucagon Epinephrine G adenylyl cyclase active inactive Phospohdiesterase cAMP ATP Glycogen Synthase active Breakdown of glycogen into glucose-1-phosphate inactive active Protein Kinase A P P inactive Stops making glycogen active Phosphorylase inactive Phosphorylase Kinase Effect in Nucleus 26 Glucagon/Epinephrine Action contd Nucleus Cytoplasm CREB plus ATP cAMP cAMP active Protein Kinase A Pepck gene CREB CRE PEPCK enzyme Starts making glucose DNA PEPCK mRNA 27 Termination of the signal
cAMP is broken down by phosphodiesterase
Phosphatase reverses the phosphorylation of the three proteins
glycogen synthase and
Adenylyl cyclase remains active while hormones are present. The cell has a way to stop adenylyl cyclase activity when it the hormones are removed.
28 Termination (the steps)
a subunit is a GTPase which hydrolyzes GTP to GDP and inactivates itself
inactive a subunit reassociates with b and g
Requires an additional factor RSG
RSG enhances GTPase and speeds up the timing step
this causes a drop in ligand concentration resulting in
dissociation and inactivation of the receptor
29 Another Means of Termination
Active inactivation of the receptor a 2 step process of desensitization. This means that the cell stops responding even when ligand is still present around the cell
1. Phosphorylation by G-protein receptor kinase (GPRK) inactivates the receptor
2. The phosphorylated receptor binds another protein called arrestin which acts as adaptor for Clathrin allowing receptors to be internalized thus further desensitising the cell
30 The Big Picture g a 2 ATP P b stops P GDP GRK 2 ADP Pi Arrestin RSG - G-protein hydrolyzes GTP inactivates itself - receptor is phosphorylated becomes inactive - phosphorylated receptor binds arrestin an adaptor for endocytosis 31 Lipid Second Mesengers 32 Lipid Second Messengers
GPRC intracellular messengers made by phosphorylation and hyrdrolysis of the membrane glycerophosphate phosphatidyl inositol
inositol sugar phosphate glycerol fatty acids - P - kinases - P - P P phospho inositol 45 biphosphate (PIP2) 33 Lipid Second Messengers
Produced by phoshatidylinositol-specific phospholipase C.
produces two signal molecules
Diacylglycerol (DAG) which stays within membrane
and Inositol triphosphate (IP3) highly soluble enters cytoplasm
P -O - P - O - O- DAG IP3 34 One Exampleacetylcholine effects on a smooth muscle cell
1. inositol phosphate is phosphorylated.
phospho inositol 45 biphosphate (PIP2)
2. acetylcholine binds to plasma membrane receptor
3. G protein system is activated (Gaq)
4. the phospholipase PI-PLCb is activated by the G protein
5. PIP2 is metabolized to diacylglycerol DAG and inositol phosphate IP3
6. DAG in turn stimulates Protein Kinase C which acts to promote muscle contractility phosphorylates elements of the actin/myosin
7. the IP3 binds to the SER membrane to IP3 receptors
8. IP3 receptors are calcium channels they release calcium from SER
9. Calcium ion concentration of cytoplasm increases
10. muscle cell contracts
11. IP3 DAG are rapidly degraded calcium is rapidly pumped back to SER
36 Phosphatidylinositol Second Messengers acetylcholine PI-PLCb G DAG PIP2 active muscle cell contraction Protein Kinase C IP3 increased Ca increased muscle contractility IP3 receptor (a ligand-gated chanel) smooth endoplasmic reticulum 37 The same basic systems are used to activate different responses to different signals
i. Vascular smooth muscle contraction
ii. smooth muscle contraction
iii. skeletal muscle contraction
iv. blood platelet aggregation of platelets
b. Protein Kinase C. cell growth differentiation (development into different tissue types) metabolism.
i. blood platelets serotonin release
ii. mast cells histamine release
iii. smooth muscle contractility
iv. nerve cells neurotransmitter release
v. adipose tissue fat synthesis
vi. liver cells glycogen hydrolysis
c. cAMP (and PKA) in different cells
i. liver epinephrine -glycogen hydrolysis glucose synthesis and glucagon -reduction in glycogen production
ii. kidney vasopressin - activation of aquaporins
iii. thyroid cell TSH - thyroid hormone release
39 The Other Receptor ClassReceptor Tyrosine Kinases (RTKs)
Receptor is an enzyme
a protein tyrosine kinase which phosphorylates proteins at tyrosines
Cell division (disorders of RTK receptors lead to uncontrolled cell division cancers).
Attachment of cells to extracellular matrix
Migration of cells
41 An Example Insulin receptor action through the RTK type receptor
Insulin is produced when blood glucose increases after eating.
It causes glucose uptake by liver muscle fat cells
Acts to decrease blood sugar cells produce glycogen or fat gluconeogenisis is inhibited.
42 Mechanism of Action
1. Receptor binds ligand on exterior of the cell.
the insulin receptor is a tetramer two extracellular alpha chains with ligand binding sites and two beta chains with kinase activity all are linked by disulfide bridges.
2. Activation of the receptor
insulin receptor is unusual in existing in dimer state even before binding the ligand
tyrosine kinase activity trans-autophosphorylati on.
it also phosphorylates another protein Insulin Receptor Substrate (IRS).
The phosphorylated IRS stays on the insulin receptor by a PTB domain
43 Mechanism of Action contd
3. Other proteins recognize bind and are activated by the phosphorylated tyrosines on receptor and IRS
4. final actions of insulin
1. PI PLCg eventually (via the second messengers IP3 Diacylglycerol Protein kinase C (PKC) and calcium described in previous lecture) causes cell proliferation
2. Protein Kinase B PKB causes
glucose uptake into cells by transferring the GLUT 4 transporter in muscle and fat cells.
increased protein synthesis
stimulation of glycogen synthase
3. Ras Promotes protein synthesis growth proliferation
44 Insulin receptor insulin 1. receptor - ligand binding binding site a a b b 2. tyrosine kinase activity - auto - kinase - Insulin receptor substrate a a b b P P P P P P P P P P IRS1 IRS1 P P 45 Insulin Receptor Action... a a b b P P P P P P P P 3. effectors with SH2 domains bind to phosphorylated tyrosines P activate PI-PLC g P IRS1 phosphatidyl inositol 3 hydroxykinase P P GDP PI3K Grb/Sos activate RAS activate Protein Kinase B GTP Glucose transport glycogen synthesis actions 46 Other RTKs the growth factors 1. some tyrosine kinases exist as monomers which come together only after binding ligand. Then they start the tyrosine kinase activity. 2. the growth factors PDGF (platelet-derived) EGF (epidermal) FGF (fibroblast). 3. These activate the MAP Kinase cascade - including a GTP binding protein Ras (Ras is a non-trimer G protein they are not called G protein coupled receptors). Ras is in active state when bound to GTP. When active it activates downstream effectors. 4. Cause tyrosine phosphoylation by tyrosine kinase located on inner membrane of the receptor protein (or in case of insulin phosphorylate the IRS). 47 Calcium as a Second Messenger 48 Calcium as second messenger
we just saw calcium acting as an intermediate of IP3 action in the cell but that is not the only way it is activated
it can also be released in response to calcium channels on the cell surface Calcium dependent Calcium release
in this case calcium is both the first and the second messenger
this accomplishes rapid amplification and rapid coordination
primary Ca channel S.E.R. Ca secondary Ca chanel Ca effect 49 Types of Calcium Responses
voltage dependent channels in nerves some muscles
receptor-mediated calcium channels
fertilization calcium channels open up in oocytes following fertilization.
prevent further sperm entry anywhere around the large cell (rapid long-distance)
begins activation of cyclins starting cell division (coordinating effect)
Plants open the outside calcium channel in response to light pressure plant hormones (abscisic acid)
calcium is stored in the vacuole of plant cells
50 calcium-induced calcium release 1. 10-3 mol/l calcium chanel 6. one effect is to promote calcium removal 4. released Ca binds to calmodulin resting ca concentration 10-7 mol/l 5. effects 2. Ryanodyne receptor 3. release smooth endoplasmic reticulum or plant cell vacuole high Ca conc. 10-3 mol/l 51 The Steps
1. Calcium ions enter cell through activated channel
2. they bind to the Ryanodyne receptor.
it does the same thing as the IP3 receptor but it is different. it does not respond to IP3
3. caclium ions released from the smooth endoplasmic reticulum (or the vacuole in plants) diffuse down concentration gradient diffuse throughout cell
4. calcium ions bind to calmodulin
5. intracellular effects
6. one of the effects is to pump calcium back out of the cytoplasm to s.e.r the plant vacuole and the exterior of cell termination of signal.
52 Why are there multiple steps
1. amplification of signal. (exponential).
e.g. one receptor can turn on several heterotrimeric G-proteins before inactivation each G-protein can activate several effector molecules before inactivating itself
a. spatial coordination throughout the cell by diffusion of second messengers.
Ca are small molecules diffuse fast works well in large cells such as muscle fertilized egg neuron
b. coordination of different responses a number of different enzymes have to work together to accomplish metabolic pathways.
53 Interconnections of signaling pathways
1. Convergence. two receptors cause the same signal to be activated.
EGF acetylcholine PIP2 PI PLCb PI PLCg IP3 DAG common effects 54 Interconnections of signaling pathways
2. Divergence. one ligand has several effects
- e.g. the insulin receptor (through IRS protein) activates
Protein kinase B (PKB)
e.g. Protein kinase A (PKA of the glucagon/epinephrine receptor) turns on three different proteins
55 Interconnections of signaling pathways
3. Crosstalk pathways interconnect (a bit of a case of convergence)
e.g. every one of the intermediate steps in the map kinase cascade can affect more than one substrate.
CREB activation (see fig 15.33)
epinephrine activates PKA which enters the nucleus when activated by epinephrine phosphorylates CREB
EGF activates Raf which activates MAP Kinase which also phosphorylates CREB
Crosstalk PKA inhibits Raf. blocking the effect of EGF on CREB
56 So What
cells can keep signals separate
second messengers diffuse rapidly but some steps including the kinases (such as PKA) are kept in certain locations in the cell.
PKA is actually held in place by special binding proteins (called AKAPs) focusing its effect in the cell.
there is evidence that the steps in the MAP Kinase cascade are kept together in one location in the cell by special scaffolding proteins limiting the location of the downstream cell response.
PowerShow.com is a leading presentation/slideshow sharing website. Whether your application is business, how-to, education, medicine, school, church, sales, marketing, online training or just for fun, PowerShow.com is a great resource. And, best of all, most of its cool features are free and easy to use.
You can use PowerShow.com to find and download example online PowerPoint ppt presentations on just about any topic you can imagine so you can learn how to improve your own slides and presentations for free. Or use it to find and download high-quality how-to PowerPoint ppt presentations with illustrated or animated slides that will teach you how to do something new, also for free. Or use it to upload your own PowerPoint slides so you can share them with your teachers, class, students, bosses, employees, customers, potential investors or the world. Or use it to create really cool photo slideshows - with 2D and 3D transitions, animation, and your choice of music - that you can share with your Facebook friends or Google+ circles. That's all free as well!
For a small fee you can get the industry's best online privacy or publicly promote your presentations and slide shows with top rankings. But aside from that it's free. We'll even convert your presentations and slide shows into the universal Flash format with all their original multimedia glory, including animation, 2D and 3D transition effects, embedded music or other audio, or even video embedded in slides. All for free. Most of the presentations and slideshows on PowerShow.com are free to view, many are even free to download. (You can choose whether to allow people to download your original PowerPoint presentations and photo slideshows for a fee or free or not at all.) Check out PowerShow.com today - for FREE. There is truly something for everyone!