Unit 3: Biosignaling

transduction

converts an extracellular signal to an intracellular response

where can you find receptors

• free floating in cytosol

• nucleus

• at cell membranes

ligand-gated channels are in either

open or close conformation

specificity

how many different ligands can bind to your receptor

sensitivity

how well the ligand binds to the receptor

• you want a low Kd

avidity

summing responses using multiple binding sites, so that when one site lets go of the ligand, the next site binds to it

amplification

when each enzyme at a step activates many more enzymes

modularity

idea that one signal can cause multiple separate responses

integration

when different signals can form a combined response or cancel each other out

“crosstalk”

3 parts of G-proteins

alpha, gamma, beta

• G-proteins bind guanine di or triphosphate (GDP or GTP)

• Gs: stimulatory

• Gi: inhibitory

glycogen breaks down into

glucose

3 types of biosignals

• autocrine

• paracrine

• endocrine

4 major types of biosignaling systems

• GPCR

• receptor tyrosine kinase (and kinase cascades)

• gated ion channel

• nuclear receptor

signal transduction/cell signaling definition

the transmission of molecular signals from the environment that are not membrane permeable to the interior of the cell

things that cells can respond to

• light

• mechanical touch

• neurotransmitters

• nutrients

• odorants

• tastants

• antigens

• growth factors

• hormones

• glycoproteins/oligosaccharides

• developmental signals

• ECM components

changes that cell signals can evoke

• differentiation and antibody production

• growth in size, shape, or strength

• gene expression

• ability to divide (sexual or asexual reproduction)

autocrine signaling

acts on the cell the produced the signal

paracrine signaling

localized signaling to a nearby cell

endocrine signaling

distant signaling through the bloodstream using hormones

the binding of a signal to the receptor causes

the receptor to change in some way, usually a shape change (induced fit occurs)

• this initiates a downstream response

if a receptor is free floating, it must be able to

get to the plasma membrane on its own

examples of each main type of receptor

GPCR: epinephrine receptor

enzyme-linked: insulin receptor

ligand-gated: nicotinic ACh receptors

nuclear: steroid receptors

other membrane: integrin receptors (for cell crawling/attachment)

the human genome has _____ GPCRs and we have _____ drugs that target them

>800

>700 drugs to target them

20% of all cancers are due to a mutation in

GPCR genes

about ____% of drugs target GPCRs

25

GPCR drugs can treat

hypertension, drug abuse, mental health, asthma, etc

types of receptors often targeted by drugs

• GPCR

• enzyme-linked

• ion channels

• others

desensitization

turning off a pathway

integration

when two signals have opposite or summing effects to create a response

divergence

when a receptor gets activated and activates two different pathways taht have different end effects

localized responseq

when the enzyme that destroys an intracellular message is clustered with the message producer, so that the message is degraded before it can diffuse to distant targets, creating a brief and local response only

typical ligands

• small ions

• organic molecules

• polysaccharides

• peptides

• proteins

what Kd values show a high affinity of receptors for their signal molecule/

10^-7 or less

cooperativity

how well binding at one site influences binding at another site

clutsering

• can use lipid rafts or maybe things are just in close proximity to speed up rxns

avidity

when one receptor releases its ligand and the next immediately binds that same ligand, combining affinity for an immediate response

every cell expresses different proteins, so

they respond to the same signal in different ways

crosstalk

integration of multiple signals

parts of a GPCR

• receptor is transmembrane and has 7 alpha helices through it

• ligand binds to the receptor

• this activates a G-protein, which regulates an enzyme

receptor tyrosine kinase

ligand binding activates a tyrosine kinase by autophosphorylation, causing a kinase cascade, eventually altering gene expression

gated ion channel

ligand goes through the channel and binds to a protein, altering gene expression

GPCRs are _____

heterotrimeric membrane-associated proteins

• have alpha, gamma, and beta subunits

• the receptor changes shape when the ligand binds

epinephrine

aka adrenaline

• made in the adrenal glands on top of the kidneys and is secreted thru the bloodstream as an endocrine signal

• mediates the stress/sympathetic response

• binds to receptors in muscle and liver cells to initiate breakdown of glycogen for energy

• also binds to receptors in adipose cells to induce lipid hydrolysis

• also binds receptors in heart cells to incr HR

which subunits of the G-protein in a GPCR are lipid anchored?

gamma and alpha

steps of GPCR activation

• ligand (like epinephrine) binds to the beta-adrenergic receptor of the GPCR

• the GPCR undergoes a shape change, and the GDP bound to the alpha subunit of the G-protein gets replaced with a GTP

• alpha separates from beta gamma and activates adenylyl cyclase (the enzyme and effector)

• adenylyl cyclase (AC) converts ATP to cAMP

• cAMP activates PKA, which phosphorylates proteins causing a cellular response

• cyclic nucleotide phosphodiesterase cleaves cAMP to form AMP so no more PKA gets activated

which two subunits of the g-protein in a GPCR are always together

beta and gamma

what causes the shape change of the alpha g-protein subunit

when GDP is exchanged for GTP

• this is what causes the alpha to dissociate from beta gamma

Galpha subunit activity

• active when GTP bound

  • the switches of the protein face in to keep the P inside

• inactive when GDP bound

  • switches face out so the P can leave the protein to bind a new GTP

  • when the P leaves it can interact with beta gamma

intrinsic rate of hydrolysis

when the last P leaves the Galpha protein after it binds a new GTP

cyclic nucleotide phosphodiesterase

enzyme that cleaves cAMP to form AMP

adenylyl cyclase (AC)

converts ATP to cAMP when activated by Galpha

cAMP is a common

secondary messenger

• causes a quick response

many GPCRs mediate their effects via

cAMP (using Gs or Gi)

cAMP _____ activates PKA (cAMP-dependent protein kinase A)

allosterically

cAMP-dependent protein kinase A

PKA

• when activated by/bound to cAMP, activates other enzymes that produce glucose from glycogen

cAMP being converted into AMP

adenosine-monophosphate

• the location of the P changes since a bond it was attached to got cleaved

what does PKA need in order to be functional

have cAMP bound to it

protein kinases

add Ps to things, typically

• Ser

• Thr

• Tyr

  • since these all have OH’s, which are easily phsophorylated

protein kinases differ in

the target proteins that they recognize

• depends on the AA sequence of the target protein that the kinases recognizes

in the cAMP pathway, cAMP molecules diffuse into the _____

cytoplasm, where they bind to and activate PKAs

• two cAMP bind to each regulatory protein of the PKA complex

• once bound, this causes a shape change so that the two catalytic proteins of the complex can dissociate from the regulatory subunits and bind to something downstream (often activates proteins to trigger breaking down of glycogen in the liver for the sympathetic/ fight or flight response

PKAs can do many different things in

diff cell types

scaffold/anchoring proteins

cluster signaling proteins together to incr the rate of rxn

• this localization of PKA via anchoring proteins, for example, allows cAMP to mediate multiple signals at once

• diff anchors are expressed in diff cell types, so changes the downstream affect of cAMP in diff cell types

amplification in GPCRs

• activation of just a few GPCRs can activate a few adenylyl cyclases which convert MANY ATP into cAMP

• but it takes two cAMP to activate one catalytic subunit of PKA

• then some PKA activates MANY kinases

• then tens of thousands of glucose molecules can eventually get released into the bloodstream from the liver

diff cells can express diff ____, which bind the same ligand/hormone

GPCRs

• hormone receptor-mediated responses regulated by G-proteins can be stimulatory or inhibitory since each hormone receptor specifically interacts with either a Gs or Gi protein

• activation of Gi proteins can reduce signaling

• basically GPCRs can use Gi or Gs proteins, therefore shutting down a pathway or stimulating one

epinephrine is a ___ signal

short-acting

• uses desensitization to turn off a pathway once it is no longer needed

  • ex: stopping glucose synthesis once the fight/flight response is no longer needed

turning off GPCR pathways via self-inactivation

• pathway could be inactivated by lowering the [ligand]

• but it is typically regulated by:

  • cyclic nucleotide phosphodiesterase converting cAMP into AMP, so there is less cAMP to activate PKAs

  • Galpha has intrinsic rate of hydrolysis, so it switches out its GTP for a GDP after a while (certain amount of time like a timer), and binding the GDP inactivates the pathway by down regulating cAMP levels since when Galpha binds GDP, it can no longer activate adenylyl cyclase to make cAMP

GEFs

guanine exchange factors

• switch GDP for a GTP

• usually activating but not always

• ex: GPCR receptor binds ligand and then changes its shape so that alpha can bind GTP (the GPCR is the GEF)

GAPs

• GTPase activating proteins

• switch GTPs for GDPs

• usually inactivating

beta-adrenergic receptors to turn off the GPCR pathway (desensitization)

• you can tell a pathway has been on bc beta and gamma G-proteins have been together for a while (so alpha must be bound to GTP and to adenylyl cyclase, keeping the pathway on) (also the ligand epinephrine is bound to the GPCR)

• so BARK (beta-adrenergic receptor kinase) gets recruited and adds a P to the GPCR, creating a binding site

• these binding sites allow beta-arrestin to bind, which initiates exocytosis at the membrane so that GPCRs are taken into the cell in a vesicle

• this decr rate of rxn for the pathway since the receptors were taken away 

agonists

structural analogs of ligands that bind to a GPCR receptor to keep the pathway going (mimics the ligand)

• usually are drugs

antagonists

structural analogs of ligands that bind to a GPCR receptor so that the ligand cannot bind (the drug has very low Kd), and inhibits the pathway

• often are drugs

morphine

an agonist of certain opioid receptors

• therefore relieves pain since it causes feelings that combat the feeling of pain

opioid receptors in the body are often

GPCRs

• GPCRs can bind to natural hormones such as endorphins and cause physiological affects like runner’s high, euphoria, respiratory depression

• happens when mu-opioid receptor (a GPCR) gets signals and then causes downstream affects that cause good feelings (which combat the feelings of pain)

what is bad about GPCR opioid receptors?

the receptors can become desensitized if they are constantly receiving ligand/drug/hormone

• this causes drug resistance and eventually addiction

second messengers

• cAMP

• IP3

• DAG (diacylgylcerol)

• hormone binds to a GPCR

• alpha activated, interacts with an effector (phospholipase C) (PLC)

• PLC’s substrate is pip2, a phospholipid

• PLC cleaves polar head groups and phosphate of the glycerophospholipid (pip2)

• the glycerophospholipid had an inositol head group, and once cleaved, it forms IP3

• the remaing glycerol backbone with two FA chains forms DAG (diacylgylcerol)

• DAG and IP3 are both secondary messengers

• DAG stays in membrane due to its FAs, it diffuses out laterally through the membrane and interacts with protein kinase C (but DAG alone cannot activate protein kinase C)

• IP3 binds to receptor-gated Ca2+ channel in ER membrane, causing gate to open and Ca2+ leaves ER

• the Ca2+ also binds protein kinase C

• now protein kinase C is fully activated

DAG and IP3 are ____ secondary messengers

lipid

• so both are membrane permeable

activated effectors often generate

secondary messengers

aspects of one pathway can influence another

crosstalk

when are G-proteins activated?

when the GDP leaves and a new GTP binds to the Galpha protein

there are many categories of

enzyme-linked membrane receptors

• can require one or two ligands

receptor tyrosine kinase activation

• ligand binds to each of the two polypeptide receptors

• the two receptors dimerize and cross phosphorylate each other at their tyrosine residues from ATP

• the phosphates on the catalytic domains can serve as docking sites for downstream proteins

  • the receptors are formed or an extracellular ligand-binding domain and an intracellular catalytic domain

in tyrosine kinases, each receptor needs its own

ligand bound 

once receptor tyrosine kinases (RTKs) are dimerized but not yet cross-phosphorylated, enzymatic activity…

incr a little bit so that they can cross-phosphorylate each other

• so most RTK catalytic domains have low intrinsic kinase activity

one activated RTK dimer can activate

10 or more different intracellular proteins simultaneously

• which can cause many diff responses

RTK pathways are especially helpful for

when a cell needs to regulate and coordinate a variety of pathways at once

IRS-1

a target protein for RTKs

• insulin-receptor substrate-1

• once the RTK gets phosphorylated, IRS-1 can bind to the P docking sites and can itself become a docking site for other proteins

the phosphorylated tyrosines of RTKs bind to ___ domains on target proteins

• SH2

• PTB (phospho-tyrosine binding) domain

• these two types of domains are domains of target proteins that bind to the Ps of the RTKs

• they have similar tertiary structure

• SH2 and PTB can couple activated RTKs to downstream components of signal pathways

proteins that only bind to RTK when the RTK is phosphorylated have certain

domains that bind to the P

• SH2 or PTB binding domains

phosphatases

remove phosphates

• the opposite of kinases

examples of some second messengers

• IP3

• DAG

• Ca2+

• cAMP

phosphodiesterases

enzymes that break down second messengers

insulin is secreted in response to 

high blood glucose

• it is released from the pancreas and goes to liver, fat, and muscle cells by traveling through the bloodstream

diabetes

the inability to make or sense insulin

• due to either a faulty receptor or an inability to produce insulin

binding of insulin to the insulin receptor causes

a cascade of events that lead to incr glu uptake and metabolism

RTKs are made up of _____ and ____ proteins

alpha and beta

• one of each

• but RTKs can vary in shape a lot

insulin receptor structure

• is an RTK and the ligand binds to one of the extracellular proteins of the receptor

• once L binds, the two intracellular proteins move closer to each other and then phosphorylate each other

• this phosphorylation at the tyrosines on the protein intracellular random coil causes the active site of the intracellular domains to open up, as the Tyr are no longer covering it

  • before the random coil with the Tyr’s was covering the active site

• the P’d Tyr’s can serve as docking sites for SH2 and PTB binding domains

Insulin pathway to signal for GLUT 4 transporter production

• insulin binds to the insulin receptor, which autophosphorylates itself at its Tyr residues

• the receptor then Ps the IRS1 on its Tyr residues, so the IRS1 basically docks into the Tyrs

• The SH2 domain of Grb2 (scaffold protein) binds to the P’d Tyr of IRS1 (so Brb2 brings IRS1 and Sos tg)

• Sos binds to Grb2 and acts as a GEF to switch out Ras’s GDP for a GTP

• so Sos activates Ras 

• activated Ras binds to Raf-1, which acts as a kinase to phosphorylate MEK on its two Ser residues

• MEK is also a kinase and Ps ERK at its Thr and Tyr

• ERK goes into the nucleus and Ps and activates a TF, Elk1, which can now associate with another TF, SRF and cause expression of GLUT4 transporter genes

____ is the point of nucleation for many other proteins in the insulin pathway

IRS1

Grb is a ____ protein

scaffold/ adaptor

• it also has a SH2 domain, allowing it to bind to IRS1

• Grb2 brings IRS1 and Sos tg