What is signal transduction?
- Biochemical mechanism responsible for transmitting extracellular signals across the plasma membrane and throughout the cell
- Changes information into a chemical signal
- Often ends with covalent (phosphorylation, adding palmitoyl) or noncovalent modification of intracellular target proteins
Outline the proteins and messengers involved in a generic signaling pathway.
- First messenger
- Receptor protein
- Upstream signaling protein
- Second messenger
- Downstream signaling protein
- Target protein
- First messengers are extracellular ligands that bind to receptor proteins
- Secondary messenger is an intracellular signaling molecule
What is a secondary messenger? What are 4 examples?
- A small (non-protein) molecule that amplifies receptor generated signal
Examples:
- cGMP (cyclic GMP)
- cAMP (cyclic AMP)
- DAG (Diacylglycerol) and IP3 (inositol 1,4,5-triphosphate)
- Ca2+
What are two major classes of receptor proteins found in Eukaryotes?
- G protein-coupled receptors
- Dissociation of heterotimeric G protein complex
- Adenylate cyclase and PLC are activated
- Receptor tyrosine kinases
- Phosphorylates Tyr residue in target protein to create docking site for intracellular signaling
How many transmembrane alpha helices does GPCR have?
7
What effect does epinephrine binding have on GPCR?
- Epinephrine (ligand), a catecholamine, binds to the receptor and leads to conformational change on cytosylic side
Outline the steps in GPCR activation
- Ligand binds to GPCR causing conformational change
- GDP-GTP exchanged and subunit dissociates
- Regulation of downstream process
Describe the structure of a G-protein
- Heterotrimeric (αβγ) membrane associated proteins that bind GTP or GDP
What does Gβγ regulate? What does Gα regulate?
Gβγ
- phospholipase A
- Ion channels
- Receptor kinases
Gα
- Activate/inhibit Adenylate cyclase
- Regulate neuronal signaling
- stimulate phospholipases
- Stimulate phosphodiesterases
Give an overview of GPCR (7 steps)
- Epinephrine binds to receptor protein
- Hormone complex causes GDP bound to Gsα to be replaced by GTP which causes Gsα to activate
- Gsα separates from Gsβγ then moves to adenylyl cyclase and activates it (may activate many other Gsα subunits)
- Adenylyl cyclase catalyzes formation of cAMP
- cAMP activates PKA
- Phosphorylation of cellular proteins by PKA causes the cellular response to epinephrine
- cAMP degrades reversing the activation of PKA
Diagram the synthesis and breakdown of cAMP
ATP –(via adenylyl cyclase)–> cAMP –(hydrolysis via cAMP phosphodiesterase)–> AMP
What does the binding of cAMP do to protein Kinase A?
- cAMP binds to R2C2 tetramere (2 regulatory subunits and 2 catalytic subunits) then catalytic and cAMP-bound R subunit dissociate
TL;DR - cAMP binding causes R subunit to dissociate, leaving the 2 now active PKA monomers
What is the difference between pseudo substrate sequence and substrate sequence in cAMP binding?
Pseudo Substrate Sequence (RRGAI)
- Fits well in active site
- Alanine cannot be phosphorylated
Substrate Sequence (RRGSI)
- Fits in active site
- Serine OH group on side chain can get phosphorylated
How does activation of protein kinase C occur?
- cAMP binds to R subunit
- R subunit dissociates causing catalytic subunit to be unblocked
What three things can result from the phosphorylation of protein kinase A?
- Phosphorylation and inhibition of glycogen impedes glycogen synthesis
- Phosphorylation and activation of enzymes involved in glycogen degradation to produce glucose
- Phosphorylation and activation of enzymes involved in gluconeogenesis
TL;DR - More glucose
How can G-protein signaling be inhibited using a GTPase activating protein?
GEF
- Guanine nucleotide exchange factor
- Promote GDP-GTP exchange
- Activate signaling
GAP
- GTPase activating protein
- Stimulates intrinsic GTP hydrolysis activity
- Inhibit signal transduction
Outline the steps of the termination of GPCR signaling.
- Ligand activates GPCR signaling
- Gβγ binding to βARK recruits kinases to GPCR cytoplasmic tail
- βARK and PKA phosphorylate serine and threonine residues in GPCR cytoplasmic tail
- β-Arrestin binds to phosphorylated GPCR cytoplasmic tail
- β-Arrestin-GPCR complex is internalized by endocytic vesicle in cytoplasm
- β-Arrestin dissociates and rebinds to another GPCR
- GPCR is dephosphorylated and either degraded (lysosome) or recycled to plasma membrane
What is a receptor tyrosine kinase and what does it do?
- Receptor that transmits extracellular signal by ligand activation of a tyrosine kinase found in cytoplasmic tail of receptor
- Activated by RTKs are dimers
- Phosphorylate downstream signaling proteins that bind to RTK phosphotyrosines
Ex. Insulin receptor
