Lecture 15: Cell communication-signalling through G-protein coupled receptors Flashcards
Why is intercellular communication necessary?
- Cellular differentiation and specialization
- Growth stimulation or termination
- Tissue formation
- Integration of metabolism
General Signaling Pathway?
What are the three types of effector proteins?
- metabolic enzyme
- transcription regulatory protein
- cytoskeletal protein
4 types of signal distribution?
- contact-dependent
- paracrine/autocrine
- synaptic
- endocrine
What is the fast effect of signaling?
- altered protein function
What is the slow effect of signaling?
- altered protein synthesis
Endocrine signals are ___ in concentration and its receptors are ____ affinity
low conc
high affinity
Synaptic signals are ___ in concentration and its receptors are ____ affinity
high conc
low affinity
Chemical types of signaling molecules
- Amino acid derivatives
- Nucleotides
- Lipids
– Steroids
– Arachidonic acid metabolites
-eicosanoids
– Retinoids
- Peptides
- Gases
– Nitric Oxide (NO)
– Carbon Monoxide (CO)
• Protons
– C. elegans muscle
Amino Acid derived signals are ____
shorter lifespan, more paracrine-like
Steroid signals are relatively ____ lasting
long
Steroid signals are derived from ____’s structure
cholesterol
Eicosanoids are derived from ___’s structure
arachodonic acid
Peptide hormones include:
- Pituitary hormones
- hypothalamic hormones
- thyroid hormones
- digestive hormones
- pancreatic hormones
Nitric Oxide is a ____
vasodilator
Nitric oxide synthase transforms ____ into NO and citroline
arginine
NO diffuses out of the ____ cell into the ____ cell
endothelial to smooth muscle
What is the name of the cytosolic intracellular receptor NO binds to?
guanyl cyclase
Membrane soluble signals (those that bind to intracellular receptors) require ____ to navigate the aqeous environment outside the cells
carrier proteins
Cell-surface receptors usually bind to ____ signals.
hydrophilic
Intracellular receptors
- Nuclear receptor superfamily
- Steroid hormone receptors are ligand-activated transcription factors
- They are modular in construction
– Ligand binding domain
– DNA-binding domain
– Variable region
• Generally function with other binding partners
What are the three domains of a steriod receptor? where are they located?
N-terminus: transcription-activating domain
Middle: DNA-binding domain
C-terminus: ligand-binding domain
Response induced by steroid hormone receptor activation
The presence of a hormone or ligand is sensed by an ____
integral membrane protein receptor
• This information is communicated across the cell membrane and transmitted by intracellular signaling molecules.
Small intracellular mediators are also referred to as ____
“second messengers”
Second messengers illicit various cellular responses:
- Activate or inhibit specific protein targets
- Increase or decrease gene transcription
3 types of cell surface receptors?
- Ion channels coupled receptors
- G-protein coupled receptors
- Enzyme-linked receptors
A nicotinic acetylcholine receptor is___
an ionictropic receptor
A muscarinic acetylcholine receptor is ____
a metabotropic receptor
Enzyme-linked receptors____
dimerize to form catalytic domains
Common Second Messangers:
- cAMP
- cGMP
- DAG (diacylglycerol)
- IP3 (inositol triphosphate)
cAMP
activates protein kinase A (PKA)
cGMP
Activates protein kinase G (PKG) and opens cation channels in rod cells
DAG
activates protein kinase c (PKC)
IP3
Opens ca2+ cannels in the ER
What are the two forms of molecular switches in singaling?
- signaling by phosphorylation
- signaling by GTP binding
A intracelllular signaling protein is ON when it’s ____ or bound to____. It’s OFF when it’s ____ or ____
ON- phosphorylated or bound to GTP
OFF- dephosphorylated or bound to GDP
protein kinases ___ a phosphate from ___
remove a phosphate from ATP and add it to the intracellular signaling protein
protein phosphatases ____ the intracellular signaling protein
dephosporylate (remove a phosphate)
GEF proteins-
-help GTP to bind by ejecting GDP
GAP
cuts of the terminal phosphate of GTP (Deposphorylates it to GDP)
Tyrosine Kinase receptors
-add phosphate from ATP to tyrosine residues
Which amino acid side chains get phosphorylated?
Tyrosine (Y), Serine (S), Threonine (T)
Signal integration
Multiple signals and their receptors can affect the same intracellular signaling molecule
What are the three types of intracellular signaling complexes?
- preformed signaling complex on a scaffold protein
- assembly of signaling complex on activated receptor
- assembly of signaling complex on phosphoinositide docking sites
What signaling pathway involves all three types of intracellular signaling complexes?
The insulin signaling complex
Receptor sequestration
Receptor down-regulation
receptor inactivation
Inactivation of signaling protein
Production of inhibitory protein
General G-protein activation
G-protein coupled receptors (GPCRs)
- Seven transmembrane domains
- Responds to very diverse ligands
– Photons
– Peptide hormones
– Eicosanoids
– Amino acid derivatives
• Largest family of cell surface receptors in eukaryotes
– >1,000 devoted to smell alone in mice
• About half of all known drugs work through Gprotein coupled receptors
For GPCRs the C-terminus is on the ____ side and the N-terminus is on the ____ side.
C=cytosolic side
N=extracellular side
The βγ subunit…
function as a unit and has never been shown to be separate in the cell.
The α-subunit is….
postranslationally modified by the addition of myristic acid to the N-terminus.
The γ-subunit is….
postranslationally modified by the addition of a polyisoprenoid, usually farnesyl.
These hydrophobic modifications (to the βγ subunit)…
serve to localize the complex to the membrane, its site of action.
the α-subunit has…
A ras domain and an AH (alpha-helical) domain
On a G-protein, the gdp binding spot is on the ___ subunit
alpha
β-adrenergic receptor is a…
G-protein coupled receptor
G-protein effectors:
- Ion channels
- Adenylyl cyclase
- Phospholipase Cβ
- cGMP Phosphodiesterase
Adenylyl cyclase produces _____
cAMP
The ___ subunit of G-protein interacts with adenylate cyclase
Adenylate kinase has ___ catalytic domains.
How does cAMP produce a cellular response?
It binds to a protein kinase (PKA)
Activated PKA bind to ____ to produce___
inactive CREB, CREB -binding protein (CBP)
CBP binds to ___
cyclic AMP response element (CRE) which activates a target gene for transcription
Acute response of cAMP
-phosphorylation of protein involves in glycogen metabolism
Delayed response of cAMP
- increase in synthesis of the somatostatin gene
Ser/Thr protein phosphatases ____ the action or protein kinases
reverse
Four types of protein phosphatases
-Type I (dephosphorylates most PKA substrates)
– Type IIA (broad specificity, main PPTase)
– Type IIB (aka Calcineurin, regulated by Ca2+)
– Type IIC (minor player)
G-protein activation of PLCβ
• Phospholipase C beta
- PLCβ is activated by specific Gα-subtypes
- Gqα, Goα, and Goβγ activate PLC
Phosphatidylinositides
- phospolipase release IP3 from glycerol back bone, forming IP3 and diacylglycerol
- diacylglycerol (DAG) activates PKC
- IP3 releases ca2+ from the ER
Calcium is sometimes used as a ____
second messenger
Positive and negative feedback produce ____
calcium waves
Ca2+ homeostasis is maintained by…
- Membrane pumps:
- Na+ driven Ca2+ exchanger
- Ca2+ Pump
What three mechanism help to maintain low cytosolic Ca2+ levels?
- Ca2+ pump in ER membrane
- Ca2+ binding molecules in the cytoplasm
- active Ca2+ import into the mitochondria
Ca2+ signaling effects are mediated by which three Ca2+ binding proteins?
- Troponin C
- Synaptotagmin
- Calmodulin
Troponin C
– Muscle contraction
Synaptotagmin
– Neurotransmission
Calmodulin
– Multipurpose Ca2+ receptor involved in many responses
– Binds 4 Ca2+ ions, need two or more or adopt an active conformation
How does Calmodulin change conformation upon Ca2+ binding?
Its two domains wrap around its target protein
What are the three types of Ca2+-Calmodulin-dependent protein kinases?
- Narrow Substrate specificity
- Myosin light chain kinase (smooth muscle contraction)
- Phosphorylase kinase (glycogen breakdown)
- Broad substrate specificity
- CaM-Kinase II
What does activated CaM kinase II do?
- Its mechanism of activation allows past calcium influxes to be “remembered”
- The enzyme also changes its activity in response to calcium oscillations
– It can react to frequency and amplitude of calcium spikes
• Important for learning an memory in the brain
_____ can sense Ca2+ oscillations
CaM kinase II
