BCMB 311- Quiz 5

Why has yeast mating served as an early model for understanding how extracellular chemical signals can be perceived and produce intracellular changes?

Yeast cells “shmoo” in response to mating factors→ form mating projection

Define autocrine signaling.

Signals act on the same cell that released them.

Define endocrine signaling.

Hormones travel long distances, typically through blood stream

Define paracrine signaling.

Signals act on nearby cells

Define contact dependent signaling.

Requires direct physical contact between cells

Yeast releasing mating factor can be classified as which type of signal?

a. Autocrine

b. Endocrine

c. Paracrine

d. Contact dependent

c. Paracrine

A variety of biomolecules can act as signaling molecules. What are they?

• Proteins

• Peptides

• Amino acids

• Nucleotides

• Steroid hormones

• Fatty acid derivatives

• Dissolved gases

What are hormones?

• A molecule produces by one organ (gland) and released into the bloodstream

• Binds to another organ (effector organ) to regulate physiology or behavior

• Ultimately, hormones regulate gene expression

What are neurotransmitters?

• A molecule released from a neuron and received by another neuron or effector organ (muscle, gland, etc.)

What is a water-soluble hormone?

• Free (unbound) in the plasma

• Usually peptides that bind to receptors on the membrane of the target cell→ stimulate the production of intracellular second messengers

• UNABLE to penetrate the cell membrane

What is a lipid-soluble hormone?

• Circulate bound to plasma proteins

• Mechanism of action is directly mediated on the nuclear chromatin→ the formation of a hormone receptor complex→ activates RNA polymerase→ new mRNA→ specific protein

• MUST cross the plasma and nuclear membranes

What two types of receptors do extracellular signal molecules bind to?

1. Cell-surface receptors

2. Intracellular receptors

What are cell-surface receptors?

Receptors on the plasma membrane that bind extracellular signal molecules and trigger the release of intracellular signaling molecules.

What are intracellular receptors?

Receptors inside the cell (inside nucleus) that bind small, hydrophobic signal molecules (usually steroid hormones)

What is signal transduction?

A stepwise progression of signaling stages: receptor→ transducers→ effectors

• The receptor receives/perceives signal

• Transducers relay signal

• Effectors convert signal to an intracellular response

What are second messengers?

Small molecules that pass along a signal initiated by the binding of a ligand (“first messenger”) to its receptor

What are the three ways receptor signals are transduced?

1. Receptor-mediated activation of phosphorylation cascades that trigger the activation of various enzymes

2. Exchange of GDP for GTP be GTP-binding proteins (G proteins), which leads to generation of second messengers (cAMP, lipid breakdown products, Ca2+, etc.)

3. Conformational changes that open ion channels or recruit proteins into nuclear transcription complexes

The same signal molecule can induce different responses in different target cells. Describe this using acetylcholine.

• Acts on heart pacemaker cells and decreases firing rate (less contractions)

• Acts on salivary gland cells and increases secretion

• Acts on skeletal muscle cells and increases contraction

How can extracellular signals act rapidly?

Can activate/deactivate an enzyme that is already present→ can be very fast

• Phosphorylation/dephosphorylation

• Ubiquitination

How can extracellular signals act slowly?

Can cause a protein to be synthesized by activating transcription→ much slower

• Can be more energetically efficient because protein is only made if needed

What roles can intracellular signaling proteins play?

1. Relay signal

2. Amplify signal (transduce)

3. Detect from multiple sources and integrate

4. Distribute signal→ creating branches in info flow

5. Feedback→ positive or negative

What is a positive feedback loop?

• Downstream effector proteins activate earlier steps→ often leads to substantial amplification

• Allows pathway to exist in a stable “on” state in the absence of receptor activation or until acted upon by separate inactivation signal

What is a negative feedback loop?

• Downstream effectors inhibit earlier steps

• Pathway will shut itself off after activating to a certain level and when receptor is no longer active

• New signal will be required to reactivate

How can some intracellular signaling proteins act as molecular switches?

1. Signaling by protein phosphorylation (activate/deactivate)

2. Signaling by GTP-binding proteins

What two types of regulatory proteins control the activity of monomeric GTPases?

1. Guanine nucleotide exchange factors (GEFs)

2. GTPase-activating proteins (GAPs)

What do GEFs do?

Promote the exchange of GDP for GTP→ switches protein on

What do GAPs do?

Stimulate the hydrolysis of GTP to GDP→ switches protein off

What are the three main classes of cell-surface receptors?

1. Ion channels

2. G-protein coupled receptors

3. Enzyme-coupled receptors

How do ion-channel-coupled receptors work?

Signal molecule binds receptor and causes a conformational change→ opens channel and allows ion flow

What are the two ways enzyme-coupled receptors work?

1. Signal molecule binds and causes inactive catalytic domains to form active dimer

2. Signal molecule binds and causes formation of dimer→ active associated enzyme can bind

All GPCRs have a similar structure. Describe this.

• Seven transmembrane a helices

• Extracellular side binds signaling molecules

• Receptors can bind:

  • Small molecules (I.e., acetylcholine and epinephrine)

  • Proteins

• Cytoplasmic side binds a G protein inside cell

• G protein→ heterotrimeric GTPase

• About 800 genes in humans encode GCPRs

What are the two types of GPCR responses?

1. Stimulatory→ Gs

2. Inhibitory→ Gi

What are the two main types of GTPases?

1. Small, monomeric GTPases (i.e., Ran)

2. G Proteins→ heterotrimeric GTPases containing a, B, and y subunits

• a subunit→ binds GTP/GDP

• By dimer

• Both (a and y) are membrane-tethered by attachment to lipid head groups

What are the typical steps involved with activating a GPCR?

1. Ligand binds extracellularly

2. Cytosolic domain undergoes conformational change→ activates a G protein

3. Activated G protein exchanges GDP for GTP in a subunit

4. Often, a subunit dissociates and binds/activates other effector proteins to activate signal transduction

5. Free By dimer can also activate effectors

What does the activated a subunit do?

• a subunit GTPase hydrolyzes its GTP to GDP (usually after a few seconds of activation)

• Inactive a subunit releases target effector protein and rebinds the By dimer→ reforms inactive G protein

• Effector protein inactivates once its no longer bound by the a subunit

What is G_i, a G protein, activation coupled to?

G_i receptor activation is directly coupled with the opening of K+ channels in the plasma membrane of heart pacemaker cells

• Activated By subunit causes opening of ion channel

Enzymes activated by G proteins ____ the concentrations of small intracellular signaling molecules called ____

increase, second messengers

• Second messengers diffuse to act on intracellular signaling proteins

1. Seven

2. Three

3. Two

4. GDP

5. GTP

6. GTPase

What is cAMP? How is it synthesized and degraded? What does it do?

• cAMP is a common second messenger (released into cytosol)

• Synthesized by adenylyl cyclase and degraded by cAMP phosphodiesterase

• Can bind and activate downstream effectors

• Synthesis/release is followed quickly by degradation/removal

What are the two main branches of the nervous system?

1. Somatic (conscious activities)

2. Autonomic (unconscious activites)

What are the two branches of the autonomic nervous system?

1. Parasympathetic (inhibitory)

2. Sympathetic (stimulatory)

What is the typical GPCR associated with the sympathetic nervous system?

G_s

• Receptors: Adrenergic receptors

• Ligand: Epinephrine

What is the typical GPCR associated with the parasympathetic nervous system?

G_i

• Receptors: Muscarinic receptors

• Ligand: Acetylcholine

How does G_i work in heart pacemaker cells?

1. Acetylcholine binds its GPCR

2. G protein activates

3. By complex opens K+ channel

4. Increases permeability to K+

5. Membrane is harder to activate→ slows heart rate

Why does epinephrine stimulate glycogen breakdown in skeletal muscle cells?

• Muscle cells need to rapidly get energy to prepare for muscle contraction

• Glycogen is a polymer of stored glucose and is present in muscle cells

• We must get to breakdown mode to generate energy in the form of ATP

What are the steps of activation for B adrenergic receptor (G_s) in skeletal muscle cells?

1. Activation of epinephrine GPCR (B adrenergic receptor) activates adenylyl cyclase

2. cAMP is produced

3. cAMP activates protein kinase A (PKA)

4. PKA has many targets→ phosphorylation by PKA typically activates target

5. Phosphorylase kinase is activated

6. Phosphorylase kinase phosphorylates and activates glycogen phosphorylase→ breaks down glycogen, releasing glucose

A rise in intracellular cAMP can _____ gene transcription. Provide an example.

activate

• Bacterial Lac operon→ expresses genes needed to catabolize lactose

How does cAMP activate PKA?

By removing inactivating “regulatory” subunits from catalytic subunits

What two signaling pathways are activated by phospholipase C?

1. IP₃

2. DAG

Some GPCRs work by activating phospholipase C. Describe this in terms of the IP₃ and DAG pathways.

• Use G protein G_q

• Both a and By are involved in activating phospholipase C

• Phospholipase C hydrolyzes an inositol head group from a membrane lipid

• Inositol 1,4,5-triphosphate (IP₃) diffuses through cytosol and opens ER Ca²⁺ channels

• Ca²⁺ floods into cytosol

• Diacylglycerol (DAG) remains in plasma membrane and, together with Ca²⁺, helps activate protein kinase C (PKC)

• PKC phosphorylates and activates downstream effectors to continue the signal

What is a second messenger?

• Molecule/ligand is “first messenger”

• Second messenger (Ca²⁺, cAMP, etc.) is released when ligand binds to extracellular receptor

• Second messenger activates/inhibits processes in cytosol or nucleus (depending on if it’s G_s or G_i)

Briefly describe the activation of phospholipase C.

• Hormone binds receptor

• G_q protein is activated

• Phospholipase C is activated

• DAG and IP₃ release from cell membrane

  • IP₃ mobilizes calcium ions (has second messenger effects) from ER (ex. smooth muscle contraction)

How are IP₃ and DAG produced?

• Many second messengers are generated by breakdown of membrane phospholipids

• Binding of some hormones to their receptors triggers activation of PLC

• Results in the breakdown of inositol phospholipids to yield IP₃ and DAG

Ca²⁺ signaling triggers many biological processes and is involved with egg fertilization. Describe this.

Fertilization of an egg by a sperm triggers an increase in cytosolic Ca²⁺ in the egg→ produces membrane changes that prevent fertilization by other sperm cells and initiates embryonic development

What does calmodulin do?

Calmodulin acts as an intermediary protein that senses calcium levels and relays signals to various calcium-sensitive enzymes

What does nitric oxide (NO) do?

NO triggers smooth muscle relaxation in blood vessel walls by bidning guanylyl cyclase and triggering the release of cyclic GMP

• Enzyme in mitochondria (mitochondrial aldehyde dehydrogenase) converts nitroglycerine into nitric oxide

How do enzyme-coupled receptors work?

• Act as enzymes themselves when activated (ex. receptor tyrosine kinase)

• Binding of signal molecule activates recpetor’s enzymatic activity

• Pass once through cell membrane (unlike GPCRs, which have 7 transmembrane configuration)

• Binding of extracellular ligand activates tyrosine kinase activity

• Results in phosphorylation of target protein in cytoplasm→ activates target protein

• Usually involves phosphorylation cascade

• All mediated by series of kinases

What three domains make up a single pass receptor tyrosine kinase and what do they do?

1. Extracellular domain→ responsible for ligand binding

2. A single transmembrane domain

3. Intracellular domain→ a tyrosine kinase: an enzyme that uses ATP to phosphorylate a tyrosine on a target protein

What is the role of kinases and phosphatases?

• Phosphorylation typically occurs on serine, threonine, and tyrosine (side chains contain -OH group)

• Phosphorylation cascade turned off by phosphatases→ remove phosphates

• Phosphorylation cascade turned on by kinases→ add phosphates

Describe the activation of RTKs.

• Ligand binding often causes receptor dimerization→ produces conformational change that activates intracellular kinase domains

• Cytosolic domains of receptor are kinases that phosphorylate each other upon dimerization

• Other proteins are recruited to phosphorylated sites and become activated→ assembly of intracellular signaling complex

Most RTKs activate the monomeric GTPase Ras. Describe this.

• Ras is tethered to the membrane via lipid attachment

• Activated RTK recruits an adaptor

• Adaptor recruits Ras-GEF to membrane

• Once enriched at the membrane, Ras-GEF activates Ras-GTP

• Ras-GTP activates a signaling cascade (MAPK)

• Ras GAP will eventually promote GTP hydrolysis to inactivate Ras until a new signaling event occurs. Phosphatases will eventually inactivate the kinases

Describe the MAP-kinase signaling module.

• Ras-GTP activates MAP kinase kinase kinase

• MAP kinase kinase kinase activates MAP kinase kinase

• MAP kinase kinase activates MAP kinase

• MAP kinase activates proteins X and Y (changes in protein activity) and transcription regulators A and B (changes in gene expression)

  • Mitogens are small proteins that promote mitosis

  • Each of these enzymes activates may targets, so each level serves as an amplification step

What is the relationship between some RTKs and PI-3 kinase?

• Some RTKs activate PI-3 kinase to produce lipid docking sites in the plasma membrane

• One downstream target of PI-3 kinase is Akt kinase

  • The IGF signal promotes cell survival/groeth through Akt

• Once the ligand dissociates, tyrosine phosphatases remove phosphates and inactivate RTK

Describe the effect of insulin-like growth factor (IGF) binding to RTK.

• IGF binds and activates RTK

• PI-3 kinase binds RTK and becomes activated

• PI-3 kinase phosphorylates/activates PIP2 (located in membrane)

• PIP2 phosphorylates/activates PIP3 (located in membrane)

• PIP3 binds/activates PK1

• PK1 and PK2 phosphorylate Akt

• Akt relays signal onward

How does Akt promote cell survival?

• Akt phosphorylates Bad of Bad/Bcl2 complex

• Releases inactive Bad and active Bcl2

• Bcl2 promotes cell survival by inhibition of apoptosis

How does the steroid hormone cortisol act?

• By activating a transcription regulator

• Steroid hormones bind nuclear hormone receptors

• Nuclear hormone receptors are intracellular receptors that also act as transcription factors to activate transcription and specific genes

• Cortisol crosses into cell through plasma membrane→ binds/activates nuclear receptor protein through conformational changes→ activated complex enters nucleus→ complex binds regulatory region of target gene→ activates transcription

How is a hormone response element activated and what does it do?

• Lipid soluble molecules (ex. steroid hormones and vitamin D) are able to penetrate cell membrane

• Bind with protein receptors in cytosol

• Hormone-receptor complex binds to a regulatory sequence of the DNA called hormone response element

• Transcription of specific gene or mRNA will be stimulated or inhibited by the hormone response element

  • Thyroid hormones have intranuclear receptors

How are estrogen receptors involved in breast tumors?

• Immunohistochemical detection of estrogen receptor complex in nuclei of tumor cells

• Breast tumor cells may or may not have estrogen receptors

• If estrogen receptors are present, there is in advantage→ hormone therapy can control tumor growth

What is EGFR and what can mutations in it cause?

• EGFR (epidermal growth factor receptor) is an RTK that normally responds to growth factors

• In some lung cancers, mutations in EFGR gene change receptor’s structure

• Mutated receptor can remain active even without growth factor binding

• This continuously activates intracellular pathways that promote cell growth and survival

What is HER2 and how is it involved with breast cancer?

• HER2 is an RTK found on cell membrane

• Normally, HER2 works to activate signaling pathways that regulate cell growth and division

• In some breast cancers, HER2 gene amplification produces too many HER2 receptors

• Excessive receptor signaling stimulates pathways that promote uncontrolled cell proliferation

  • Clinical connection: HER2-targeted drugs (ex. Herceptin) can block HER2 signaling and help treat HER2-positive breast cancer

When adrenaline binds to adrenergic receptors on the surface of a muscle cell, it activates a G protein, initiating an intracellular signaling pathway in which the activated α subunit activates adenylyl cyclase, thereby increasing cAMP levels in the cell. The cAMP molecules then activate a cAMP-dependent kinase (PKA) that, in turn, activates enzymes that result in the breakdown of muscle glycogen, thus lowering glycogen levels. You obtain muscle cells that are defective in various components of the signaling pathway. Referring to the Figure provided, indicate how glycogen levels would be affected in the presence of adrenaline in the following cells. Would they be higher or lower than in normal cells treated with adrenaline?

A. cells that lack adenylyl cyclase

B. cells that lack the GPCR

C. cells that lack cAMP phosphodiesterase

D. cells that have an α subunit that cannot hydrolyze GTP

A: Higher

B: Higher

C: Lower

D: Lower

What are the three types of protein filaments? Order them from largest to smallest.

Microtubules> Intermediate filaments> Actin filaments

Describe the structure of microtubules.

• Hollow tubes of 25 nm thickness

• Made of a-tubulin and B-tubulin monomers, with B being a GTPase

• Rigid but do not have high tensile strength

• Has polarity→ plus and minus end

• Minus end usually attaches to the microtubule organizing center (MTOC) in animal cells

What are the main functions of microtubules?

• Organization within cytosol

• Tracks for transport of vesicles and organelles

• Movement of chromosomes during cell division

• Serves as moving structural component within cilia and flagella

• Provide structure for:

  • Mitotic spindle

  • Cilia and flagella

  • Centrioles

  • Neurons

What is the role of tubulin and protofilaments in microtubule structure?

• a and B tubulin form a tubulin dimer

• A linear strand of dimers is called a protofilament

• A ring of protofilaments makes up a microtubule

• All dimers and protofilaments assemble in the same orientation

Describe the polarity of microtubules.

• a tubulin is exposed at the minus end

• B tubulin is exposed at the plus end

• Polarity is critical for directional transport of cargo along microtubules

• Microtubules grow faster from their plus ends

Tubulin polymerizes from ____ sites on a _____. Can a and B tubulin spontaneously come together in vitro?

nucleation, centrosome

• In vitro, a and B tubulin can spontaneously come together as rings of protofilaments, self-assembling into microtubules→ only at a concentration far higher than is present in the cell

What does it mean for microtubules to have a property known as dynamic instability?

Each microtubule grows and shrinks independently of its neighbors

What controls the dynamic instability of micro tubules? What favors assembly/disassembly? What does “catastrophe” refer to?

GTP hydrolysis:

• B tubulin is a GTPase→ hydrolyzes its GTP over time

• GTP-bound tubulin dimers favor assembly

• GDP-bound tubulin dimers favor disassembly

• “Catastrophe”→ loss of GTP tubulin cap and rapid depolymerization

How are microtubules stabilized? How do they behave when stabilized/unstabilized?

• Can be stabilized by attachment to capping proteins

• Unstabilized→ microtubules grow and shrink randomly

• Stabilized→ microtubule plus ends that encounter a stabilizing capping protein will persist

  • Microtubules dynamically grow out from centrosomes in all directions and retract (disassemble), probing cell for attachment sites that will stabilize ends in places where microtubules are needed

How do microtubule-affecting drugs act?

• Can bind and stabilize microtubules

  • Taxol→ anti-breast cancer

• Can bind tubulin dimers and prevent polymerization

  • Colchicine, colcemid

  • Vinblastine, vincristine→ Hodgkin’s disease and Wilms tumor

What are the two motor proteins discussed? In what direction do they move and what are their functions?

• Dynein

  • Retrograde: from + end to - end

  • Function: cilia motion

• Kinesin

  • Anterograde: from - end to + end

  • Function: cell division and intracellular trafficking

• Walk in a directional fashion

• Require ATP

In a ciliated cell, how does a cilium “beat?” What is the role of basal bodies?

• Cilium beats by performing a repetitive cycle of movements→ power stroke (whipping motion) followed by a recovery stroke

• Basal bodies act as MTOC to stabilize minus ends

How do flagella propel a cell through fluid?

By using repetitive wavelike motion

What is the general structure of cilia and flagella? What is their role in the trachea, fallopian tubes, and sperm?

• Microtubule-rich organelles

• Structure: 9 + 2 arrangement of MT doublets

  • Trachea: moving mucus and dirt

  • Fallopian tubes: moving ovum from ovary to uterus

  • Sperm: motility

What is the result of dynein movement in isolated doublet microtubules vs. normal flagella? Where do immobilized dyneins try to “walk?”

• Isolated doublet microtubules (no linking proteins)→ dynein produces microtubule sliding

• Normal flagella (have linking proteins)→ dynein causes microtubule bending

• Immobilized dynein→ try to walk toward minus end of other microtubule doublet

What causes Kartagener’s syndrome? What are the symptoms?

• Mutation in dynein motor arm

• Results in immotile cilia and sperm (flagella)

• Symptoms:

  • Male and female infertility

  • Recurrent sinusitis

  • Bronchiectasis

  • May result in situs inversus→ inverse arrangement of internal organs

How are microtubules involved in transport along a nerve cell axon?

• Microtubules guide the transport of organelles, vesicles, and macromolecules in both directions along a cell axon

  • Backward transport (to cell body)

  • Outward transport (to axon terminal)

  • Recall that centrosomes are usually near the nucleus such that the plus ends radiate out

What are the steps of kinesin movement?

1. Kinesins (with bound cargo) randomly bind to a microtubule

2. The leading head group exchanges its ADP for ATP→ triggers conformational change

3. Linker neck region of leading head group “zippers” onto head group→ throws lagging head group forward one “step”→ called power stroke

4. Trailing head group hydrolyzes ATP and linker region releases

5. Leading head group exchanges ADP for ATP→ linker neck region zippers→ process repeates

Describe the structure of actin filaments.

• 7 nm diameter (smaller than MTs 25 nm)

• Twisted chain of identical actin monomers

• Has polarity (+/- end)

• Nucleation, dynamics, and branching heavily regulated by binding proteins

• Most abundant near cell cortex

What are the main functions of actin filaments?

• Vesicle and organelle trafficking

• Help determine cell morphology and dynamics:

  • Microvilli

  • Phagocytosis

  • Cell constriction during cytokinesis

  • Cell motility by crawling along surfaces

• Alongside bundles of myosin motor proteins→ make up contractile component of muscles

• Provide mechanical strength to plasma membrane

What are 4 shapes/functions of actin filaments?

1. Microvilli (in intestinal cells)→ not motile like cilia, increase surface area for absorption

2. Contractile bundles→ generate contractile forces for cell shape, adhesion, migration, mechanical stress resistance, etc.

3. Filopodia→ protrusions used to reach out and adhere to surfaces as part of cell crawling

4. Contractile ring→ used during cytokinesis

What is the shape of actin filaments?

Two strands of identical actin monomers in a right-handed helix

What type of enzyme do actin monomers act as?

ATPases (B-tubulin of MTs=GTPase)

Where do actin monomers add to form the filament?

Actin monomers can add to either end, but addition to plus end is more favorable→ occurs at lower actin-ATP concentrations

Actin filaments can undergo treadmilling. Describe this.

Treadmilling→ occurs when monomers are added to the plus end and are eventually removed from the minus end

Can microtubules undergo treadmilling?

Yes→ tubulin is added to plus end and lost from minus end

What 3 drugs are discussed that act on actin filaments? How do they do this?

1. Phalloidin→ binds and stabilizes filaments

2. Cytochalasin→ Caps filament plus ends, preventing polymerization there

3. Latrunculin→ Binds actin monomers and prevents polymerization

________ ______ control the behavior of actin filaments in vertebrate cells.

Actin-binding proteins

What percent of cellular protein is actin? How much is in filaments vs. monomers?

• About 5%

• Half in filaments

• Half are monomeric