BMED 5207 exam 4

general principles of signal transduction

extracellular signal molecules as a key mediator of cell communication; receptor proteins transmit signals from extracellular stimuli; activated receptors relay signals through intracellular signaling proteins; regulation of signal transduction-signal amplification and repression

common techniques used for studying signal transduction

how to characterize the ligand/receptor interaction; how to detect signaling status; how to detect molecular interactions; how to detect transcriptional response

simple signal transduction pathway

extracellular signal molecule interacts with receptor protein allows for cascade of intracellular signaling proteins that interact with effector proteins

Types of cellular stimuli that can initate intracellular signaling

physical stimuli, chemicals or small molecules, soluble peptides and proteins, tethered molecules and proteins

examples of physical stimuli that can initiate intracellular signaling

light, temperature, mechanical force

examples of chemicals or small molecules that can initate intracellular signaling

amino acids, lipids, steroids, acetylcholine, oxygen, nitric oxide

examples of soluble peptides and proteins that can initiate intracellular signaling

insuling, growth hormone, many hormones

examples of tethered molecules and proteins that can initiate intracellular signaling

extracellualr matrix, cell surface proteins

Ways that extracellular signal molecules can bind receptors

contact dependent, paracrine/autocrine, synaptic, endocrine

Paracrine

sends molecules to nearby cells

autocrine

sends molecules to act on itself

What are the different effects that different combinations of extracellular stimuli can cause?

survive, grow and divide, differentiate, die

extracellular stimuli on different cells

effects are cell-type dependent

acetylcholine on heart muscle cell, skeletal, and salivary gland

heart: decreased rate and force of contraction; skeletal: contraction; salivary: secretion

types of receptors

cell-surface receptors and intracellular receptors

where do signal molecules bind cell surface receptors?

hydrophilic or hydrophobic signal molecule will bind to a receptor on the plasma membrane

types of intracellular receptors and how signal molecules interact with receptor

nuclear receptors and cytosolic receptors; small hydrophobic signal molecules are transported into the cell with a carrier protein to interact with the receptor

cell surface receptors types

ion-channel-coupled receptors; G-protein-coupled receptors, enzyme-coupled receptors

Nuclear receptors

are transcription factors; contain 2 major domains - DNA binding and transcription binding domain; interact with coactivtor and inhibitory proteins

whan can intracellular second messengers do?

transduce a signal; concentrations of these change following ligand binding; act as ligands for intracellular protein/enzymes

intracellular second messenger(s) used by G protein-coupld signaling pathways

cAMP, DAG, IP3

intracellular second messenger(s) used by nitric oxide receptor and receptor guanylate cyclase

cGMP

What is a common second messenger?

calcium

What are many effectors activated by?

phosphorylation or GTP binding

What do phosphorylated proteins act as?

molecular switches to transmit a signal

What amino acid is phosphorylated to modify activity of target protein in phosphorylated proteins?

tyrosine or serine/threonine

Phosphorylated protein receptor relationship with kinases

some have intrinsic kinase activity, others associate with kinases in the kinases in the cytosol or in the membrane

Phosphorylated protein receptor kinase activity can be modulated by what?

other kinases, binding to other proteins, second messenger levels

Phosphatases regulate what?

the phosphorylation state o fphosphorylated protein receptors (dephosphorylate)

What other reversible post-translational modifications (PTM) act as molecular switches for signaling (besides for phosphorylation)?

acetylation, methylation, ubiquitination

What can GTP bound proteins act as?

molecular switches to transmit a signal

What is bound to the GTP binding proteins to allow for interaction with effector proteins to modulate thier activity?

GTP

What do GEFs promote in GTP-bound proteins?

release of GDP

What act as GEFs for heterotrimetric G proteins?

GPCR

What do GAPs promote in GTP binding proteins?

hydrolysis of GTP

What are GAPs besides from the inactivator protein for GTP proteins?

transducer proteins that pass the signal to the next step in a pathway

What do scaffold proteins do?

regulate the speed, efficiency and specificity of signaling

types of scaffold proteins

preformed signaling complex on a scaffold protein, assembly of signaling complex on an activated receptor, assembly of signaling complex on phosphoinositide docking sites

What does formation of a signaling complex use?

SH domains (SH2 and SH3) and PTB domain

what does SH domain stand for?

Src Homology domain

What does PTB domain stand for?

Protein Tyrosine Binding domain

Insulin receptor example of molecular interaction domains

IRS docking protein is recurited to plasma membrane, which contains a PTB domain that binds to receptor and the receptor phosphorylates IRS; phosphorylated IRS recruits Grb2, which contains a SH2 domain that binds IRS1 and a SH3 that recognize polypropylene

Positive feedback regulation of signaling

length of time the signal is on is enhanced

Negative feedback regulation of signaling - short delay

signal is turned off or dampened

negative feedback regulation of signaling - long delay

signal oscillates

adaptation of signaling-desensitization

receptor sequestration, receptor down-regulation, receptor inactivation, inactivation of signaling protein, production of inhibitory protein

Characterization of the receptor/ligand interaction

receptor binding assays and functional responses

Receptor binding assays

quantify the number of receptors on the membrane (Bmax) and determine the affinity their interaction with a ligand (kd)

Receptor/ligand interaction

is reversible, equilibrium is reached when association = dissociation, equilibrium is not a static point but one where the rate of change in both directions is balanced

Receptor/ligand interaction: rate of association

[R] [L] k_on

Receptor/ligand interaction: rate of dissociation

[RL] k_off

What happens when a ligand is added ot a system (association/dissociation)?

association > dissociation

What happens when a ligand is removed form a system (association/dissociation)?

dissociation > association

How to perform receptor binding assaays?

incubate a series of concentrations of a labeled ligand with cells containing the receptor, for determining total binding; incubate a series of concentrations of a labled ligand in the presence of saturating concentration of unlabeled ligand with cells containing the receptor, for determining nonspecific binding; after binding , separation of the ligand bound receptor from the free ligand - vacuum filtration; measurement of the ligand bound receptor complex (e.g. by scintillation counter for radioactivity); generate a binding curve with specific binding data and te concentratron of ligand

K_d

the concentration of ligand where 50% of the receptor binding sites are occupied, k_off/k_on

What makes a ligand “high affinity”?

k_on > k_off; K_d is very small; the smaller the K_d the higher the affinity

How can a ligand be classified as (based on the cellular response of the ligand binding to the receptor)?

agonist and antagonist

agonist

activate cellular response

antagonist

no cellular response and inhibit the response to an agonist

what is the activity of an agonist measured by?

the concentration of ligand that elicits 50% of maximal cellular response (EC50)

activation of few than 100% of receptors usually elicits what?

near-maximal celluar response (EC50 < K_d)

how to detect signaling status?

IP and western of post modifications and second messenger assays

Detecting signaming status: PTMs

detecting protein phosphorylation through metabolic labeling 32P, detections with generic phosphorylation antibodies; detection by phospho-specific antibody

Detecting signaling status: second messenger assays

calcium indicators: fura-2 binds to calcium, calcium concentration affects the wavelength at which fura-2 is maximally ecited

detecting molecular interactions

FRET, Co-Immunoprecipitations

what can FRET monitor?

activation-dependent interactions in cells

Co-immunopreciptiations

isolate cellular complexes, dependent on antibodies

How do Co-IPs differ from normal IP?

with respect to lysis conditions

Detecting transcriptional responses

transcripitional activation can be measured by luciferase assay, assay using transient transfection of reporters require normalization

importance of GPCRs

~800 GPCRs encoded by ~4% of human genes; virtually all physiological functions are GPCR dependent; three of five senses are mediated by GPCRs; estimates are that ~33% of all FDA-approved drugs act on GPCRs; over ~100 orphan GPCRs and ~134 of GPCRs are targetd dby clinically useful drugs

the receptor signaling system

ligands, receptor, g proteins, regulators, effectors

Structure of G protein linked receptors

seven transmembrane alpha helical loops, four extracellular segments, four cytosolic segments; oreintation always conserved; cytosolic segments interact with G proteins and determine signaling specificity

GPCR ligand binding sites

within a hydrophobic core, on exterior surface, extended N-terminal

example(s) of ligands that can bind within hydrophobic core of GPCRs

biological amines, light or ordorants, peptides or chemokines, purines, lipids

example(s) of ligands that can bind on exterior surface of GPCRs

peptides

example(s) of ligands that can bind extended N-terminal

biological amines, glutamate, Ca2+

GPCRs activate effectors through

heterotrimeric G proteins

GTPyS and GMPPNP

non-hydrolysable analogs of GTP can be used to force extended signaling activity

Which subunits of G proteins activate downstream effectors?

both free G_a and G_By

Heterotrimeric G proteins consist of multiple what?

alpha, beta and gamma subunits that are rapidly dissociated by activated receptors

How are the subunits of G proteins tethered to the membrane?

Ga and Gy subunits are covalently attached lipids and oftened localized to lipid rafts

How many different subunits of G proteins are there?

21 G_a, 6 G_B, 12 G_y

Different G_By subunits

have not been found to differ functionally

Different receptors interacting with specific Ga subunits

activate specific signaling pathways; defined by which alpha subunit is present in the associaed hetero-trimeric G protein

How are GPCRs classified?

based on their G protein coupling

Ga classes

Gi-coupled, Gs-coupled, Gq-coupled, G12/13-coupled

what are cAMP efectes mainly mediated by?

protein kinase A (PKA)

cAMP acting on PKA

inactive PKA is a hetero-tetramer of two regulatory and two catalytic subunits; binding of first cAMP to a regulatory subunit lowers Kd for binding of the second (cooperative binding); cAMP binding to regulatory subunits releases active catalytic subunits

PKA in different cell types

acts on different substrates; PKA phosphorylates a serine or threonine resides within the common sequence motif: X-R-R (K)-X-Ser/The-Φ (hydrophobic)

PKA can activate transcription of what?

CREB repsonse genes

How does PKA activate transcription of CREB responsive genes?

activated PKA can translocate into nucleus and phosphorylate CREB; activated CREB binds to the CRE sequence to regulate transcription of target genes

Amplification of an extracellular signal by what?

cAMP and PKA mediated signal transduction

What is PKA activity localized and antagnonized by?

localized by AKAP and antagonized by PDE

AKAP

a kinase-associated protein family of PKA anchoring proteins; localize effects of cAMP-PKA to specific regions of the cell

PKA-PDE negative feedback loop

provides close local conrol of the cAMP level and PKA activity; AKAP anchors PKA regulartory subunit and cAMP PDE to the nuclear membrane; hormone activation of GPCRs increases local cAMP to a level in escess that can be degraded by basal PDE; cAMP binding to the PKA R subunits releases active catalytic subunits into th ecytosol; some C subunits enter the nucleus and phosphorylate active CREB to turn on expression fo CRE-genes; some C subunits phosphorylates acitve PDE to hydrolyze cAMP back to basal levels, inactivating PKA; PDE dephosphorylation by phosphatases returns the complex to the resting stage

Desensitization or adaptation can take place where?

at the level of the receptor or downstream

What can down-regulate GPCR signaling?

phosphorylation and arrestin binding

arrestin

binds to phosphorylated GPCR to allow for endocytosis (phosphorylation on intracellular loop 3)

Modulation of GPCR signaling by RGS

the activated GTP-bound Ga subunit returns to the inactivated GDP-bound G_aBy complex by hydrolyzing GTP to GDP

what is the hydrolysis of GTP to GDP mediated by in GPCR signaling?

by the intrinsic GTPase activity of the Ga, which is accelerated by the GTPase accelerating protein (GAP), RGS (regulator of G protein signaling)