Biochem exam 4

What are the general components of signaling pathways

What are the general components of signaling pathways

First messenger, receptor protein, upstream signaling, secondary messengers, downstream signaling proteins, and target proteins

Signal transduction

the biochemical mechanism responsible for transmitting extracellular signals across the plasma membrane and throughout the cell

What does signal transduction often end with

covalent or noncovalent modification of intracellular target proteins

what are the most common covalent protein modifications

phosphorylation and dephosphorylation

what are the three consequences of activating a receptor protein

covalent protein modification

protein conformational changes

altered rates of protein expression

What are first messengers

Extracellular ligands that bind to the receptor proteins

What are secondary messengers

intracellular signaling proteins

how do you determine upstream/downstream

before the 2nd messenger is upstream, after the 2nd messenger is downstream

paracrine

signaling between different types of adjacent cells

autocrine

cell signaling between same type of cells

Hormones

biologically active compounds that are released into the circulatory systems and come into contact with hormone receptors in the target cells

First messengers and hormones can be categorized as what

Endocrine, paracrine, or autocrine

What are the steps when NO is the first messenger

Arginine (produces NO) undergoes NO synthase to become Citrulline

NO activates Guanylate cyclase (upstream signaling protein)

Guanylate cyclase is phosphorylated from GTP, resulting in PPi and cGMP

cGMP PDE can be inhibited by sildenafil (is a competitive inhibitor)

cGMP can activate protein kinase G resulting in vasodilation

Where is NO synthesized

in endothelial cells

Can NO pass through the cell membrane

yes, it is small enough to pass through

what are secondary messengers

Small, nonprotein intracellular molecules that amplify receptor-generated signals

What are some examples of secondary messengers

Cyclic GMP (cGMP)

cAMP

Diacylglycerol (DAG)

Inositol-1,4,5-triphosphate (IP3)

Ca2+

How do you turn PIP2 into IP3 and DAG

Add PIP2 to water, and phospholipase C (PLC) breaks it down to result in IP3 and DAG

What are the two results from the phosphoinositide cascade

phosphorylation of downstream target proteins or Ca2+ -calmodulin binding to downstream target proteins

What is the pathway of the phosphoinositide cascade that ends with Ca2+ -calmodulin binding to downstream target proteins

Receptor activation activates PLC (phospholipase C)

PLC breaks down PIP2 to DAG and IP3

IP3 activates the Ca2+ channels in the endoplasmic reticulum

Ca2+ second messenger

Ca2+ -calmodulin binds to downstream protein

What is the pathway of phosphoinositide cascade that ends with phosphorylation of downstream target proteins (2 ways)

Receptor activation of PLC (phospholipase C)

PIP2 is broken down by PLC into IP3 and DAG

1. IP3

IP3 activates Ca2+ channels in the endoplasmic reticulum

Ca2+ is second messenger

Ca2+ activates PKC (phosphokinase C) to phosphorylate downstream target proteins

2. DAG

DAG is second messenger

DAG activates protein kinase C to phosphorylate downstream target proteins

what are the five classes of receptor proteins in Eukaryotes

G protein-coupled receptors

Receptor tyrosine kinases

Tumor necrosis factor receptors

Nuclear receptors

Ligand-gated ion channels

What is the function of G protein-coupled receptors

Dissociation of heterotrimeric G protein complex

Activation of adenylate cyclase and PLC

ex. Epinephrine, glucose, smell, taste, vision

What is the function of receptor tyrosine kinases

phosphorylates Tyr residues in target proteins to create docking sites for intracellular signaling

(no secondary messengers)

what is the function of tumor necrosis factor receptors

Transmit extracellular signals by forming receptor trimers

controls inflammation and apoptosis

What is the function of nuclear receptors

modulate gene expression through protein-DNA and protein-protein interactions

What is the function of ligand-gated ion channels

control the flow of K+, Na+, and Ca2+ ions across the cell membrane in response to ligand binding

Which sensory perceptions are involved with G protein-coupled receptors

vision, taste, and smell

How many alpha helices are involved in G protein-coupled receptor signalling

Seven transmembrane alpha helices

What is another name for G protein-coupled receptors

serpentine receptors

Which is attached to the extracellular domain in G protein-coupled receptors

Glycoproteins that contain carbohydrate functional groups

what is the Beta2-adrenergic receptor (a G protein-coupled receptor)

It is adrenal based

Epinephrine (ligand), a catecholamine, binds to the receptor and leads to a conformational change

what is a receptor agonist

they activate receptor signaling by mimicking the natural ligand

What are receptor antagonists

they bind to the receptors with high affinity and block the binding of physiological agonists without promoting structural changes needed for signal transduction

ex. of physiologic adrenergic receptor agonists

epinephrine, norepinephrine, and dopamine

Pharmaceutical adrenergic receptor agonists

clonidine and isoproterenol

pharmaceutical adrenergic receptor antagonists

metoprolol and prazosin

different G protein-coupled receptor-mediated signals can be integrated within what

a single cell type in response to multiple extracellular stimuli

What type of enzyme does glucagon and glucagon receptors utilize

adenylate cyclase

what type of enzyme does epinephrine and Beta 2-adrenergic receptors utilize

adenylate cyclase

what type of enzyme do epinephrine and alpha1-adrenergic receptors utilize

phospholipase C

what is the secondary messenger for glucagon and glucagon receptors

cAMP

what is the secondary messenger in epinephrine and beta2-adrenergic receptors utilize

cAMP

what is the secondary messenger in epinephrine and alpha1-adrenergic receptors utilize

DAG, IP3

what type of signaling does glucagon and glucagon receptors use

G(salpha)

what type of signaling does epinephrine and beta2-adrenergic receptors use

G(salpha)

what kind of signaling does epinephrine and alpha1-adrenergic receptors utilize

G(qalpha)

what are some signaling functions of the alpha subunit of GPCRs

Activate adenylate cyclase

inhibit adenylate cyclase

regulate neuronal signaling

stimulate phospholipases

stimulate phosphodiesterases

what are the steps in GPCR activation

Ligand-induced conformational changes in the GPCR

Receptor-mediated stimulation of guanine nucleotide exchange (GTP replaces GDP)

Regulation of downstream effector processes by G(alpha)-GTP and G(beta/gamma) complexes

Termination of signal

what is the general mechanism for the visual pathway

Rhodopsin stimulates OMP inducing a conformational change

Conformational change activates G(alpha)

G(alpha) with GTP stimulates PDE (phosphodiesterase)

PDE converts cGMP to GMP

What is the general mechanism of taste

Taste molecule binds to taste receptor which induces a conformational change of OMP

Conformational change activates G(alpha)

G(alpha) with GTP activates PLC (phospholipase C)

PLC breaks down PIP2

PIP2 is broken down into IP3 and DAG

What is the main mechanism for the olfactory pathway

Olfactory molecule binds to an olfactory receptor which produces a conformational change in OMP

conformational change activates G(alpha)

G(alpha) with GTP activates AC (Adenylate cyclase)

AC breaks down ATP into PPi and cAMP

when is protein kinase A (PKA) active

when cAMP levels are high

What are the three distinct metabolic responses from protein kinase A (PKA)

Phosphorylation and inhibition of glycogen synthase(opposite of glycogen phosphorylase); impedes glycogen synthesis

Phosphorylation and activation of enzymes involved in glycogen degradation to produce glucose

Phosphorylation and activation of enzymes involved in gluconeogenesis

when is glucagon released

when not eating

When is epinephrine released

fight or flight

what is the net affect for glucagon and epinephrine pathways

net glucose export

Describe the shared pathways between glucagon and epinephrine

Glucagon binds to glucagon (or epinephrine binds to a beta 2) receptor, induces a conformational change, G(salpha)dissociates once GTP is present, G(salpha) activates AC which causes ATP to be broken down to PPi and cAMP, which stimulates PKA for net glucose export

Describe the parallel pathways of epinephrine

Epinephrine binds to a B2 receptor which stimulates G(salpha) with GTP to activate AC breaking down ATP into PPi and cAMP which activates PKA for net glucose export

Epinephrine binds to alpha1 receptor which stimulates G(qalpha) with GTP to activate PLC breaking PIP2 down into IP3 and DAG, both of which stimulate net glucose export

what are the two results from activating PKA

regulatory subunit dimer and two active PKA monomers

How is PKA activated

Inactive catalytic subunit (PKA) is bound to the regulatory subunit (R)

cAMP is a regulatory molecule that, when bound to R allows R and PKA to dissociate to (R/cAMP and PKA monomers)

PKAs role in epinephrine (B2 bound) and glucagon pathway

Activation of glucagon or B2 receptor activates G(alpha) to activate AC, breaking down ATP to PPi and cAMP

cAMP is the second messenger that activates PKA

PKA has a side reaction that binds with cAMP phosphodiesterase generating AMP

PKA has 3 results all of which stimulate net glucose export

PKA results (turn off glycogen synthesis, turn on glycogen degradation, turn on glucose synthesis(gluconeogenesis in the liver))

PKAs role in epinephrine (alpha1 bound) pathway

activation of alpha1-adrenergic receptor activates G(alpha), which activates PLC breaking down PIP2 to DAG and IP3

DAG activates PKC which turns off glycogen synthesis for net glucose production

IP3-mediated calcium release from the ER (activates Ca2+) either turning on glycogen degradation or turn off glycogen synthesis leading to net glucose production

GEF

Guanine nucleotide exchange factor

promotes GDP-GTP exchange

activates signaling

GAP

GTPase Activating Proteins

Stimulate intrinsic GTP hydrolysis activity

Inhibit signal

Describe the G protein cycle

(GPCR are controlled by GEFs and GAPs)

Ligand stimulation of the GEF function of a GPCR

Dissociation of G(alpha) and G(beta/gamma) from a GPCR

GAPs such as RGS2 stimulate the intrinsic GTPase activity of G(alpha)

Reassociation of G(alpha/beta/gamma) with a GPCR

what are 2 ways to terminate GPCR mediated signaling

Desensitize the receptor after G(alpha/beta/gamma) dissociation

G protein-coupled receptor kinases (GRK) phosphorylate the GPCR cytoplasmic domain on Ser and Thr residues

Beta-adrenergic receptor Kinase (BARK) pathway steps

Ligand activation of GPCR signaling

G(beta/gamma) binding to BARK recruits this kinase to the GPCR cytoplasmic tail

BARK and PKA phosphorylate Ser and Thr residues in the GPCR cytoplasmic tail

Beta-Arestin binds to the phosphorylated GPCR cytoplasmic tail

Beta-Arestin-GPCR complex is internalized by endocytic vesicles in the cytoplasm

Beta-Arestin dissociates and can rebind another GPCR

GPCRs are phosphorylated and either degraded in lysosomes or recycled to the plasma membrane

what is receptor tyrosine kinase (RTK) signaling

Transmits extracellular signals by ligand activation of an intrinsic tyrosine kinase (phosphorylates itself on tyrosine residues) function found in the cytoplasmic tail of the receptors

Phosphorylate downstream signaling proteins that bind to RTK phosphotyrosines

What are activated receptor tyrosine kinases called

Dimers

What are targets of RTK (receptor tyrosine kinases)

adaptor proteins (which act as molecular bridges that facilitate signal transduction)

Kinases

what are examples of RTK

Epidermal growth factor (EGF)

Insulin

What is the general pathway for growth factors binding to RTKs

Ligand binding, receptor dimerization, and kinase activation

Phosphorylation of RTK cytoplasmic tails

Protein binding to RTK phosphotyrosines and phosphorylation of target proteins

Activation of downstream signaling pathways

What is the two step model in a serum growth factor

Ligand (EGF) binds to two EGFR to induce receptor dimerization, which leads to kinase activity of one EGFR (EGFR1) and phosphorylation of Tyr residues on the other EGFR (EGFR2)

Phosphorylation on EGFR2 induces conformational change and phosphorylates Tyr on EGFR1

EGF: a serum growth factor

binds to the EGFR and stimulates receptor dimerization on the cell surface

EGF cell signaling pathway steps

(unaliganded EGFRs with unphosphorylated C-terminal Tyr residues)

EGF binding reduces dimerization and EGFR1 phosphorylation of EGFR2 Tyr residues

Conformational change leads to EGFR2 phosphorylation of EGFR1 Tyr residues

(binding of SH2 domain proteins and initiation of downstream signaling)

EGF mechanism

EGFR dimer is activated

Adaptor protein (GRB2) with 3 domains binds to GEF protein

Adaptor protein domains (SH2/SH3/SH3) Sh2 look for phosphorylated tyrosine, SH3 rich in prolines and looks for GEF protein

GEF protein (SOS son of sethernless) GEEF exchange GTP for GDP and binds to GDP and RAS (G protein)

G protein is functionally the same as G(alpha)

RasGAP (activates intrinsic ATPase of Ras (puts it inactive)) stimulates GTPase in Ras to deactivate Ras signaling

Activation of downstream signaling pathways

Ras proteins

Member of G protein signaling molecules (G(alpha))

Activated by GEFs

Anchored to the cytoplasmic side of plasma membrane and covalently attached to the lipid

contain an intrinsic GTPase activity and are regulated by GAPs

Ras protein signaling steps

Recruitment of RAF to the membrane by Ras-GTP

Src phosphorylation of Raf (to activate Raf)

Raf phosphorylation of MEK (Raf is a kinase)

MEK phosphorylation of ERK

Nuclear translocation of phosphorylated ERK (Phosphorylated ERK homodimer is located inside the cell, increase rate of cell division and phosphorylation)

regulation of gene expression by ERK phosphorylation of transcription factor proteins

(end result is increased rates of cell division)

Describe how Ras is the most common mutation with oncogenes

An oncogene won’t activate because it will always have GTP cells to continue to proliferate

different mutations are responsible for different cancers

Inactive wild-type Ras protein is defective and becomes an activated mutant Ras protein (defective GTPase)

This activates MEK leading to proliferation even in the absence of EGF hormone

insulin signaling

insulin binds to the insulin receptor and induces a conformational change

insulin contains two chains, which are held together by disulfide bonds

insulin signaling pathway

Insulin is a growth pathway (has the same first steps)

Ras attaches to SOS and GRB2 but this attaches to Shc by phosphorylation and PTB domain is phosphorylated to B1

Tyrosine autophosphorylation leading to recruitment and phosphorylation of substrates

Insulin only one needed to activate

PIP3 activation and insulin signaling steps

Insulin receptor-mediated activation of PI3K through phosphorylation of IRS

PI3K phosphorylates PIP2 to form PIP3

Phosphoinositide-dependent kinase-1 phosphorylates Akt

Glucose uptake and glycogen synthesis

Glucose uptake pathway

IRS-1, phosphorylated by the insulin receptor, activates PI-3K by binding to its SH2 domain. PI-3k converts PIP2 to PIP3

PKB bound to PIP3 is phosphorylated by PDK1 (not shown). Thus activated PKB phosphorylates GSK3 of a Ser residue inactivating it

GSK3 inactivated by phosphorylation, cannot convert glycogen synthase (GS) to its inactive form by phosphorylation, so GS remains active

Synthesis of glycogen from glucose is accelerated

PKB stimulates movement of glucose transporter GLUT4 from internal membrane vesicles to the plasma membrane, increasing the uptake of glucose

Tumor necrosis factor receptor signaling

a single receptor stimulates intracellular pathways with opposing cellular responses

What does activation of TNF receptor complex

Binding of TNF-alpha induces a conformational change in the TNF receptor at the death domain

This causes a silencing of the death domain

TNF receptor-associated death domain (TRADD)

TNF receptor signaling is inhibited by SODD proteins (silence of the death domains)

TNF-alpha binding stimulates the exchange of SODDs for TRADDs

TRADD proteins initiate adaptor complex formation and downstream signaling

Apoptosis steps (apoptotic pathway)

TNF receptor-mediated assembly of DD and DED protein complexes

Autocleavage of procaspase 8

CASP8 cleavage of procaspase 3

CASP3 cleavage of cellular proteins

Apoptosis steps (cell survival pathway)

NIK/RIP-mediated phosphorylation of IKK

IKK phosphorylation of IkBalpha results in activation of p50/p65

Active p50/p65 heterodimeric NFkB translocases to the nucleus

Increased expression of anti-apoptotic genes that inhibit CASP8 and CASP3 activation

Nuclear receptor signaling

Also known as intracellular receptors, not bound to membrane, serves as transcription factors that regulate gene expression

Examples of nuclear receptor signaling

steroid receptors (nonpolar and can diffuse through cell membrane)

metabolite receptors

Nuclear receptor signaling is governed by what three parameters

Cell-specific expression of nuclear receptors

localized bioavailability of ligands

differential accessibility of target gene DNA sequences in chromatin to nuclear receptor binding

Steroid receptors have what

inverted repeat DNA sequences

Metabolite receptors have what

direct repeat DNA sequences

examples of steroid receptors

glucocorticoid receptor

estrogen receptor

Androgen receptor

Progesterone receptor

Aldosterone receptor

Examples of metabolite receptors

Retinoid X receptor (RXR)

vitamin D receptor

Retinoic acid receptor

Thyroid hormone receptor

peroxisome proliferator-activated receptors

Metabolite receptors

forms head-to-tail heterodimer

bind to direct DNA sequences that is, 5’-AGGTCA-3’

Ligands are derived from vitamins, unsaturated fatty acids, essential amino acids

Glucocorticoids are important for what

Lung development, carbohydrate metabolism, and the inflammatory response

Glucocorticoid signaling pathway

Glucocorticoids bind to the ligand-activated receptor, (heat shock proteins can stabilize the inactive complex until it becomes active)

Ligand-activated GR migrates from the cytosol to the nucleus where it activates the Annexin I gene or inactive complex

The annexin I gene reduces inflammation, increased expression of this gene leads to reduced inflammation

The inactive complex is formed from the NFkB heterodimer which inactivates the Ligand-activated complex.

The inactive complex is only present when annexin I is not present. The decrease in heterodimer (because one domain is binding to the ligand-activated complex) leads to a decreased expression of inflammation leading to a net reduced inflammation (COX-2 is what gets targeted by anti-inflammatory drugs)

Metabolism

the collection of biochemical reactions in a free-living organism that converts chemical energy into work and is divided into catabolism and anabolism.