Cell Signalling Flashcards
Signal Transduction
- conversion of one signal into another
- uses growth factors, cytokines, hormones
Elements of Cell Signalling
1) Signal or Signalling Molecules – ligand
2) Receptors – cell-surface or intracellular
3) Intracellular Signalling and Effector Proteins
4) Secondary Messengers – amplify signal
Nuclear-Receptor Superfamily
- intracellular receptor = interact w/ hydrophobic proteins
- estrogen, progesterone, glucocorticoid, thyroxine, retinoic acid receptors
- all receptors have –> ligand and DNA binding domain and variable region
Gene Activation by Nuclear Receptor
no glucocorticoid –> inhibitor (Hsp90) bind to ligand binding domain
glucocorticoid –> inhibitor released from LBD
- glucocorticoid receptor moves into nucleus
- bind to promoter elements in DNA
- activation domain brings TF’s
Forms of Intracellular Signalling
Endocrine Signalling
- tissue produce ligand + hormone secretion DISTANT
Paracrine Signalling
- cell produce ligand + acts on ADJACENT cell
Autocrine Signalling
- cell produce ligand + it’s receptor = self-stimulate
Signalling by PM
- cell produce TM protein + interact with adjacent cell receptor
Signalling by Cell-Surface Receptors
1) synthesis + release of signalling molecule
2) bind to specific receptor = conformation change
3) intracellular signal-transduction pathways
4) short term cellular responses
- cell metabolism/function/movement
5) long term cellular responses
- gene expression/development
5) terminate cellular responses (feedback)
Dissociation Constant
- measure of affinity of a receptor and ligand
- ligand concentration to bind 50% cell receptor
- lower Kd = lower ligand concentration = high affinity
Kd= [R][L]/[RL]
Functional Expression Assay
Identify cDNA encoding cell surface receptors
- culture cells do not express receptor for ligand X
- transfect w/ cDNA library + screen cellular phenotype
- identify added cDNA by PCR ==> sequencing
Regulation of Protein Activity
Kinase
- activated by signalling –> add phosphate = ACTIVE
Phosphatase
- activated by signalling –> lose phosphate = INACTIVE
G-Protein Coupled Receptor System
- a receptor has 7 membrane-spanning domain
- a coupled trimeric G protein
- a membrane-bound effector protein
G-Protein Complex
Trimeric G Protein
- Gα – lipid anchored at cytoplasmic face (GDP/GTP)
- Gγ – lipid anchored at cytoplasmic face
- Gβ – linked to Gγ
- transmit signal from G-protein coupled receptor to effector proteins
G-Protein Coupled Receptor Pathway
1) ligand bind to GPCR
2) recruit Gα subunit + trimeric complex
3) conformational change – dissociate GDP
4) bind GTP to Gα
5) conformational change – dissociate receptor + Gβγ
6) Gα binds + activate effector protein
7) GTP hydrolysis = Gα dissociate from effector and bind to Gβγ
FRET and Trimeric Complex
Inactive Receptor + Trimeric Complex
- excite CFP (Gα) release wavelength excite YFP (Gβγ)
ADD LIGAND…
Active Receptor + Gβγ + Gα
- excite CFP (Gα) release wavelength = cyan
- no interaction
Effector Proteins
Adenylyl cyclase –> 2nd messenger = cAMP
Phospholipase C –> 2nd messenger = IP3 or DAG
Activation of Muscarinic Acetylcholine Receptor
- acetylcholine bind to its receptor
- opening of K+ channel and cell polarization
- Gβγ subunit activates effector protein (K+ channel)
Regulation of Adenylyl Cyclase
Stimulatory Hormones (epinephrine, glucagon)
- binds to receptor –> stimulatory G protein complex
- activate adenylyl cyclase = form cAMP
Inhibitory Hormones (PGE1 and adenosine)
- binds to receptor –> inhibitory G protein complex
- inhibit adenylyl cyclase = no cAMP
Cholera Toxin
Inappropriate activation of adenylyl cyclase
- toxin enters via ganglioside receptor in small intestine
- toxin maintain in active state
- mass activation of adenylyl cyclase
- mass increase in cAMP
- hyperactive CFTR ion channel
- Cl- pumped out AND Na+ and H2O follows = diarrhea
Pertussis Toxin
Whooping Cough
- toxin enter ciliated epithelial cells in lungs
- toxin inhibits inhibitor
- mass activation of adenylyl cyclase
- mass increase in cAMP
- hyperactive CFTR ion channel
- Cl- pumped out AND Na+ and H2O follows = mucous
Intracellular Second Messengers
cAMP – activates protein kinase A
cGMP – activates protein kinase G
DAG – activates protein kinase C
IP3 – opens Ca2+ channels in ER
Protein Kinase A (PKA) Activation
cAMP binds to regulatory subunits
- binding 1st cAMP to CNB-B lowers Kd for binding of 2nd cAMP to CNB-A
- catalytic domain separates after binding
Regulation of Glycogen Metabolism
- high cAMP levels = active protein kinase A
Activating phosphorylation
- active glycogen enzymes = glycogen –> glucose
Inhibitory Phosphorylation
- inhibit glycogen synthesis = NO glucose –> glycogen
- inhibit phosphoprotein phosphatase
- — activate inhibitor = inactive (phosphorylated)
Regulation of Glycogen Metabolism
- low cAMP levels = active protein kinase A
Inhibitory Phosphorylation
- inactive glycogen enzymes = NO glycogen –> glucose
Activating Phosphorylation
- active glycogen synthesis
Activation of Gene Transcription by G-Protein Coupled Receptor
- genes regulated by protein kinase A have specific nucleotide sequence = cAMP response element (CRE)
- activation of CRE-binding protein’s TFs by phosphorylation
CRE-Binding Protein Signalling Pathway
- G-protein activation –> adenylyl cyclase activated
- increase cAMP levels –> activate protein kinase A
- catalytic subunits of PKA translocate to nucleus
- phosphorylate + activate CRE-binding TF
- associates with co-activator CBP/P300
- bind to CRE = activate transcription
