-
What makes up the cell membrane?
- Phospolipid bilayer
- Proteins
- Cholesterol (animals)
-
What kinds of proteins make up the cell membrane?
- Peripheral
- Integral (Transmembrane)
-
What part of a protein is glycosylated?
The portion facing the extracellular matrix
-
What are the functions of the membrane?
- Selective barrier
- In eukaryotes, formation of organelles/compartments
- Localization of enzymatic reactions
- Cell-cell communication
- Transmission/reception of signals
- Shape
- Receive stimuli
- Site of ECM attachment
-
Where does glycoslyation occur, and what is the destination?
- Golgi
- Cell membrane (i.e. not organelles)
-
What does flippase do?
It "evens out" enzymes since most are on cytosol side
-
What is a lipid raft?
A "microdomain" of the plasma membrane which aggregates proteins and phospholpids for transportation.
-
How does a lipid raft travel?
Via vesicle
-
Do proteins for organelles get glycosylated? Why or why not?
- No.
- Glycosylation is for cell-cell recognition
-
What is an advantage of a compartment/organelle?
- Useful for specialized enzymatic reactions.
- Greater rate of collisions for reactions to occur.
-
What is the minimum length of a transmembrane protein?
20 a.a.'s
-
What are some functions of transmembrane proteins on the cytoplasmic side?
- Intrinsic or associated enzymatic activity
- Provide cell with shape
-
What are functions of transmembrane proteins on the outside?
- Receptors for soluble ligands
- Channel/gate
- ECM attachment (e.g. integrins)
- Cell-cell attachment (e.g. cadherins)
-
How are gates/channels activated?
Hormones or action potentials
-
What are types of secondary structures for transmembrane proteins?
- Alpha helices
- Beta barrels
-
For alpha helices in a gate, where is the hydrophobic side?
Facing the outside surface of the gate
-
What transports water thru the membrane?
Aquaporin
-
What is the function of membrane cholesterol?
Provides membrane fluidity
-
What types of molecular movement are there in the membrane?
- Rotation on axis
- Lateral (sideways)
- Flip-flopping (from one side to the other)
-
Describe flip-flopping movement in a membrane.
- Requires energy and is thus uncommon
- Requires the flippase enzyme
- Proteins are too large for this movement
-
What can cross a membrane by simple diffusion?
CO2 and O2
-
What happens if a cell membrane has no fluidity?
The cell dies
-
What are some ways to alter membrane fluidity?
- Change length of hydrocarbon tails
- Change saturation
- Change cholesterol content
- Change temperature
-
What does an increase in cholesterol do?
Increases fluidity
-
What does an increase in temp do?
Increases fluidity
-
What does an increase in saturation do?
Decreases fluidity
-
What does an increase in hydrocarbon tail length do?
Decreases fluidity
-
What is a ligand?
A signal that is received by the cell usually resulting in a response.
-
What is a receptor?
A feature on the cell which receives a ligand
-
How does a receptor respond?
- Intrinsically as an enzyme.
- Associated with an enzyme.
- With the cytoskeleton directly.
-
What are some responses to a ligand being received?
- Gene expression
- Metabolism
- Movement
-
What are effector proteins?
They are proteins activated by intracellular signalling proteins
-
What are three types of effector proteins?
- Metabolic enzyme
- Gene regulatory protein
- Cytoskeletal protein (altered cell shape or movement)
-
What types of interactions are there between effectors and cells?
- Direct contact between transmembrane molecules of two neighboring cells
- Paracrine system (local)
- Synaptic (neurotransmitters)
- Cytonemes (thin cytoplasmic extensions releasing hormones)
- Endocrine (via bloodstream)
-
Which type of signalling requires the most signal molecules?
Endocrine
-
Which type of signalling is not soluble?
- Contact dependent
- Maybe Cytonemes?
-
What are three types of signalling responses?
- Quick; < 1 hour; protein de/activation; synaptic
- Slow; 18-24 hours; transcription/translation; endocrine
- Single cell or group in development; autocrine
-
What are four end-results of a cell in response to a signal?
- Survive
- Grow + Divide
- Differentiate
- Die
-
Describe how acetylcholine can cause two different reponses.
- Heart muscle - decreased rate and force of contraction
- Skeletal muscle - contraction
-
What are two types of signals wrt to water?
- Soluble - interact with transmembrane receptors
- Insoluble - interact with cytoplasmic receptors
-
Non-soluble signals affect ___.
transcription
-
What are examples of non-soluble signals?
Vitamin D, estrogen, testosterone, cortisol, estradiol, retinoic acid, and thhroxine
-
Non-soluble signals have a ___ effect due to ___.
- longer-lasting
- their duration in the blood
-
Non-soluble signals enter the cytoplasm by ___.
diffusion
-
Non-soluble receptor responses include ___.
- Early - first 30 min
- Delyated
- Depends on type of protein and timing of synthesis
-
Gene expression regulation falls into two general categories:
-
All nuclear receptors bind as either ___ or ___.
Homodimers, heterodimers
-
An inactive receptor protein is bound to ___ proteins.
Inhibitory
-
Proteins in the primary response can ____.
Activate other proteins for a delayed/secondary response.
-
Describe responses to testosterone.
- Early - male characteristics in development
- Delayed - muscle growth
-
Describe responses to estrogen.
- Early - female characteristics
- Delayed - retention of bone mass
-
What are three classes of cell-surface receptors?
- Ion-channel-coupled (open channel)
- G-protein-coupled (G activates enzyme)
- Enzyme-coupled (intrinsic and associated enzymatic)
-
What are three types of "players" that affect the cell?
- First messengers - ligands such as hormones
- Intracellular signaling proteins
- Second messengers - not unique to one pathway
-
What are examples of second messengers?
cAMP, cGMP, 1,2-diacylglycerol (DAG), IP3, Ca+2
-
What second messengers require ATP?
cAMP, cGMP
-
What second messengers are derived from phospholipids
DAG, IP3
-
Name various proteins/molecules in signalling pathways/cascades.
Anchoring, amplifier, integrator, modular, relay, scaffold, transducer
-
Describe an anchoring protein.
Anchors proteins to a structure at a precise location where needed.
-
Describe amplifier proteins.
Greatly increases signal they receive.
-
Describe Integrator proteins.
Combine signals from two or more pathways before forwarding.
-
Describe modular proteins.
Modify the activity of signaling proteins to regulate signal strength.
-
Describe relay proteins.
Pass messages to the next signaling component in the pathway.
-
Describe scaffold proteins.
Bind to multiple signaling proteins together in a functional complex for quicker and more efficient interaction.
-
Describe transducer proteins.
Convert singal to a different form.
-
Describe the structure of a G Protein-linked receptor (or G protein-coupled).
- Seven transmembrane spanning domains.
- Large cytoplasmic region that associates/activates with trimeric G proteins
-
Does the GPCR have intrinsic enzymatic activity?
No.
-
What are 4 classic downstream targets of G Proteins that regulate different effectors?
- Adenylyl cyclases
- Phospholipases
- Ion channels
- Protein kinases
-
What are the basic subunits of a G protein?
alpha, beta, gamma
-
What about G proteins might explain highly specific responses?
Different isoforms
-
What part of the G protein binds with GDP?
The alpha subunit
-
When activated, the beta/gamma complex can ___.
move and activate other targets.
-
What else provides specific signaling specifity?
- Cell-specific receptors, G isoforms, and effectors
- Amount of receptors, G isoforms, and effectors
- Organization of signaling cascades
- Accessory proteins
-
How do accessory proteins regulate G protein action?
They regulate the strength, efficiency, and specificity of the transmitted signal.
-
What are some examples of accessory proteins?
- GAP-43 - promotes GDP dissociation
- AGS3 - stabilizes G-alpha-GDP interaction
- Tubulin - directly transfers GTP to G-alpha
-
What are three types of accessory proteins?
- Activators of G protein signaling (AGS)
- Regulator of G protein signaling (RGS)
- Inhibitors of GDP dissociation
-
Describe activators of G protein signaling.
Can activate G proteins without the use of a receptor
-
Describe regulators of G protein signaling.
- Accelerate the GTPase activity of specific G-alpha subunits.
- Quick inactivation - hydrolysis
-
What is the typical end of a pathway?
Cell division
-
What are other roles associated with G proteins?
- Golgi stability (alternative binding partners)
- Cell polarity in the fruit fly and nematode
- Neurite outgrowth and path-finding
-
What do G protein-linked receptors activate?
- Adenylyl cyclase
- Phospholipase C-beta
-
What effect does the activation of adenylyl cyclase typically have?
- Increase of cyclic AMP concentration in the cytosol.
- This rise activates PKA.
- PKA enters the nucleus and phosphorylates CREB.
- CREB recruits CBP, and both stimulate gene transcription.
-
What is produced from the hydrolysis of PIP2?
- inositol 1,4,5-trisphosphate (IP3) - releases Ca2+ from the ER
- diacylglycerol (DAG) - helps to activate PKC
-
Describe how GPCRs increase cytosolic Ca2+ and activate PKC.
- PLC-beta is activated by G protein (via alpha, beta/gamma, or both).
- Two messenger molecules, P3 and DAG, produced from hydrolysis of PIP2.
- IP3 releases Ca2+ from ER
- Ca2+ and DAG activate PKC
-
What other purpose does the release of Ca2+ serve?
Prevents polyspermy by creating fertilization envelope
-
In the notch pathway, ___ and Jagged are ___.
Delta, ligands
-
Notch is composed of ___.
- NECD - Notch extracellular domain
- transmembrane domain
- NID - Notch intracellular domain
-
___ cleaves the NECD from the ___.
ADAM TM-NICD
-
What is ADAM?
A desintegrin and metalloprotease
-
The NICD makes its way to the ___ and generally results in ___.
- nucleus
- transcription, cell division, differentiation
-
Hh are ___, a ___.
- ligands
- family of secreted proteins
-
Hh generally functions in ___.
development
-
In adult cells Hh can lead to ___.
cancer
-
Hh homologues in vertebrates include ___.
sonic, desert, Indian Hh
-
What is the receptor for Hh?
Patch
-
What are the receptors for Hh in mammals?
-
Besides Patch and Hip, what else is needed for Hh pathway activation?
Smo
-
The ultimate target of Hh in the fruitfly is ___, a ___.
- cubitus interruptus (Ci)
- transcription factor
-
What vertebrate homologue most closely resembles Drosophila Hh?
Desert
-
How do Shh, Dhh, and Ihh differ?
Typically by potency - Shh>Ihh>Dhh
-
What role in develoment does Hh play?
cellular proliferation, growth, and axon path finding
-
What are examples of human developmental disorders from Hh?
- Holoprosencephaly
- Greig cephalopolysyndactyly syndrome
- Pallister-Hall Syndrome
- Gorlin's syndrome
-
What are some cancers triggered by malfunctioning Hh?
- Basal cell carcinoma
- Rhabomyosarcoma
- Medulloblastoma
- Small cell lung cncer
- Pancreatic cancer
-
What are some components of the Hh pathway?
- Patch(Ptc) - membrane receptor
- Smoothened (Smo) - intermembrane protein
- Intracellular Hh Signaling complex (HSC)
-
Describe Patch.
- Membrane receptor which activates Smo when bound to Hh.
- After binding, Ptc levels decrease as a result of endocytosis
-
In vertebrates, Ptc does not have ___, so it needs ___.
-
Describe Smoothened.
Intermembrane protein that when activated relays signals to HSC
-
In vertebrates, Smo is always ___.
at the cell membrane
-
Describe HSC.
- Coastal 2 (Co2) - kinesin-related protein
- Fused (Fu) - Ser/Threo kinase
- Supressor of fused (Su/Fu)
- Cubitus Interruptus (Ci)
-
What are three Ci homologs as activators in mammals?
Gli1, Gli2, Gli3
-
Since Gli is acts only as an activator, it does not get ___.
truncated
-
What happens to Ci when there is no Hh?
HSC truncates Ci which becomes a repressor
-
What happens when Hh binds to Ptc?
Production of Ci which becomes an activator
-
In the Drosophila wing imaginal disc, Ci is truncated where?
In all but cell fate 1 nearest the posterior
-
In the Drosophila wing imaginal disc, Hh concentration results in activation where?
In all but cell fate 5 (nearest the Anterior) which results in repressor.
-
In the Drosophila wing imaginal disc, how does Hh concentration affect expression?
[Hh] is proportional to activation
-
What is involved in Hh processing?
- Autocleavage
- Binding of cholesterol to C end
- Addition of palmitate to amino terminus
-
Describe the binding of Hh to cholesterol.
- Critical for target cell intake
- Critical for signal transduction after Hh binds to Ptc
- If binding is inhibited, Hh doesn't work
-
In the absence of Hh, Ptc ___.
blocks the phosphorylation and stability of Smo.
-
What type of receptor does Smoothened have?
G protein coupled receptor
-
When there is no Ptc, ___.
Smo is found in endosomal vesicles
-
Upon Hh binding to Ptc, ___.
Smo is released and localizes to the cell membrane
-
Smo multimers may be required for ___.
high level signaling
-
With Hh present, Ci__ is processed to ___.
- 155
- Weak activator - Ci^act
- Strong activator - Ci*
-
If no Hh is present, Ci__ is formed.
75
-
How is Ci phosphorylated?
- PKA
- Glycogen synthase Kinase 3-beta
- Casein Kinase 1-gamma
-
Upon phosphorylation, Ci is ___ by ___, a ___.
- ubiquitinated
- Slimb (supernumerary limbs
- proteosome for cleavage
-
Describe Coastal 2 (Cos2)
- Possibly a microtubule-motor
- Interacts with Smo
- Responsible for moving Smo
-
How do Cos2 and Smo interact?
- Cos2 binds Fu to Smo
- Their interaction is critical for hh pathway signalling
-
Where does Cos2 move Smo?
- cell membrane upon Hh pathway activation.
- Intracellular vessicles in the absence of Hh ligand
-
Describe Fused (Fu)
- Has kinase activity, i.e. might phosphorylate Cos2 and Su(Fu) upon Hh pathway activation.
- Binds to Cos2 and Su(Fu) via carboxy terminus domain
-
Describe Suppressor of Fused
- May be antagonistic to Fu
- No a.a. homology to known proteins
- Binds Fu and Ci, but not Cos2
-
What evidence is there that Su(Fu) and Fu might be antagonistic?
Su(Fu)- and Fu- flies yield a wt phenotype
-
What are possible functions of Su(Fu)?
- May inhibit Ci activation
- Nuclear translocation of Ci
- Transcriptional regulation in vertebrates
-
Describe Hh pathway at no/low [Hh].
- Ptc on cell membrane repressing/sequestering Smo at vesicle with HSC-A inactive.
- HSC-R on MT picking up vesicle leading to truncated Ci^75 by Su(Fu).
- Ci^75 to nucleus as repressor
-
Describe Hh pathway at medium [Hh].
- Hh binds Ptc at cell membrane permitting Smo/HSC-A to go to cell membrane
- HSC-R not at MT
- HSC-A allows Ci^act into Nucleus for low activation with possible low amount of Ci^75.
-
Describe Hh pathway at high [Hh].
- Hh sequesters Ptc to vesicle permitting Smo Multimer/HSC-A to go to cell membrane
- Fu phosphorylates Cos2/SuFu
- SuFu leaves HSC
- Cos2 drives Ci to Smo
- HSC-R not present/inactivated.
- HSC-A allows Ci* to be processed untruncated into Nucleus by SuFu for high activation
-
Research shows that Hh signalling malfuction is responsible for:
- Formation of tumors
- Survival of tumors
-
In mammals, absence of Hh leads to ___.
- Gli forms MT-attached complex with Fu and SuFu
- Gli remains in cytoplasm
-
In mammals, if Hh is present, ___.
- Hh binds to Ptc
- Smo is activated (no longer supressed)
- Processing of Gli is activated
- Gli is translocated to nucleus
-
In mammals, the negative feedback regulators in the Hh pathway are ___.
Ptch, Hip, Gli
-
What genes are for cell proliferation?
- Cyclins D1 and D2 (mammalian) -> mitosis
- cMyc
-
What are three target proteins?
- Cyclin B - Mitotic P Factor (MPF) - nuclear translocation
- P21 inhibition - tumor supression
- PDGF pathway activation (MAPK) - cell division
-
What are two types of genetic problems w.r.t. cancer?
- LOF for tumor supression - Ptc and SuFu
- Overespression of oncogenes - Shh and Smo
-
LOF of SuFu leads to ___.
medulloblastoma
-
LOF of Ptc1 leads to ___.
cell nevus carcinoma
-
Heterozygous Ptc mice ___.
develop tumors
-
Blocking Smo blocks ___.
binding of Hh
-
What should be inhibited for cancer treatment?
Smo, Gli
-
What are inhibitors of Smo?
- Cyclopamine - binds to Smo, but difficult to synthesize and toxic
- KAAD - modified cyclopamine - less toxic
-
How do you inhibit Gli?
- Forskolin - PKA activator - used in different pathways as well
- RNAi - Not feasible for treatment
-
In connective tissue, the main stress-bearing component is the ___.
ECM
-
In epithelial tissue, the ___ form the main stress-bearing component.
cytoskeletons of the cells themselves (linked by anchoring junctions)
-
What is the purpose of the ECM?
- Provides scaffolding and support for tissues and cells.
- Signal transduction.
-
What makes up the ECM?
proteoglycans, collagen, laminim, fibronectin, and vitronectin.
-
Describe proteoglycans?
"filler" substance. Traps water. Binds cations.
-
Describe collagen?
Most abundant ECM component. Connective tissue.
-
Describe laminin?
Forms network of weblike structures that resist tensile forces.
-
Describe fibronectin?
Glycoproteins. Maintains cell shape.
-
Describe vitronectin.
Glycoprotein. Promotes cell adhesion and spreading.
-
What are two principles of tissue formation?
- Cells must be attached to each other.
- Cells must be attached to a scaffold.
-
Why must cells be attached to each other?
Protein-protein interaction between cells.
-
Why are cells attached to a scaffold?
- Cells secrete proteins and carbs which make up the ECM.
- Intermembrane proteins connect the ECM with the cell's cytoskeleton.
-
What types of proteins are used in cell adhesion?
Cadherins, selectins, integrins, Ig family
-
Describe cadherins.
Tissue specific; dimerize; Ca+2 dependent; regulate cell shape and migration.
-
Cancer cells also change ___ expression.
cadherin
-
Cadherins affect cell shape and migration via ___.
Indirect binding
-
Describe selectins.
- Cell adhesion molecules that bind to sugars.
- Type of lectin.
-
In wound-clotting, what is selectin-dependent?
Weak adhesion and rolling.
-
In wound-clotting, what is integrin-dependent?
- strong adhesion and emigration
- Lets white blood cells exit capillary
-
What are three kinds of cell junctions?
-
Describe adhesive junctions.
- Desmosomes and adherens.
- Hold cells together in fixed positions w/in tissues.
- Ca+2 dependent.
-
What are two types of adhesive junctions?
Desmosomes and adherens (both Ca+2 dependent)
-
Describe the structure of desmosomes.
- Keratin intermediate filaments connected to plaque.
- Plaque composed of anchor proteins.
- Transmembrane cadherin adhesion proteins attached to plaque.
-
What makes up the transmembrane cadherin adhesion proteins?
desmoglein and desmocollin.
-
What proteins make up the plaque?
- desmoplakin
- plakoglobin
- plakophilin
-
What is the purpose of the intermediate filaments attached to the plaque?
structural support (not movement)
-
Describe tight junctions.
- Seal space between cells.
- Prevent flow of molecules and ions thru EC space.
- Important for organs that store liquids.
-
What proteins make up tight junctions?
claudin and occluding
-
Describe gap junctions.
- Most common type of junction between animal cells.
- Form open channels between cells allowing ions and small molecules to pass.
- Useful for cell-cell communication.
- Open at low Ca+2 and low pH
-
What binds to gap junction to open the channel?
calmodulin (also binds to calcium)
-
What kind of molecule can pass thru a gap junction?
small (e.g. cAMP)
-
How is the ECM formed?
Secreted by the cells
-
Describe integrins.
- Used in cell-cell adhesion.
- Serve as attachment to ECM.
- Bind to specific ECM proteins.
-
Describe how collagen fibers are formed.
- Procollagen triple-helix formed in ER/Golgi complex.
- Single procollagen molecule out via secretory vessicle.
- Cleavage of propeptides.
- Thousands of collagen molecules form fibril in ECM.
- Aggregation of fibrils form collagen fiber.
-
What is the purpose of proteoglycans?
- Trap water and provide elasticity (e.g. skin).
- "Filler" substance.
- Hold ECM in place.
-
Describe structure of proteoglycans.
- 95% carbs by weight.
- glycosaminoglycan (GAG) is main component.
- Single polypeptide with hundreds of GAGs.
-
What holds the ECM in place?
Linkages of proteoglycans to cell membranes.
-
What are three types of interactions for proteoglycans?
- Receptors.
- Binding to ECM.
- Integrins binding to proeins in ECM.
-
What are two adhesive glycoproteins?
fibronectin and laminin
-
What's the main purpose of Fibronectin?
Provides/maintains cell shape.
-
Describe fibronectin structure.
- Two large polypeptides (not identical) linked by disulfide bridges.
- Some domains bind to ECM.
- Other domains bind to membrane receptors.
-
How is fibronectin specificity determined?
By the a.a.'s flanking the RGD motif
-
What are integrins?
- Receptors that mediate attachment between cells and ECM/other cells.
- Critical for growth, hemostasis, and host defense.
- Interact with cytoskelton.
-
Describe integrin structure.
- Heterodimeric with alpha and beta subunits.
- Variable subunits - mammals have 18 alpha, 8 beta
- e.g. melanoma: alpha-v, beta-3
-
Describe the integrin receptor.
- Binds to soluble and attached ligands.
- Binding changes conformation of the dimer.
- Binding is Mn+2 dependent.
- Clustering occurs with other integrin receptors upon ligand binding.
-
What are the two types of integrin activation?
- Outside-in - info from outside to cell
- Inside-out - info from cell to outside
-
Integrins can bind to the ___, with a ___ of integrins.
same target, cluster
-
What is anoikis?
Cells cease to be bound to ECM
-
What are the typical results of integrin signalling?
Cell death, cell migration, cell shape change, cell division
-
What does integrin clustering do in normal cells?
Affects cell migration and differentiation.
-
What does integrin clustering do in cancer cells?
- Angiogenesis and metastasis
- Focal adhestion tyrosine kinase -> cell survival
- MAP Kinase -> differentiation, cell growth, apoptosis
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