Lecture 3 Flashcards
Describe two different experiments to determine if a hormone acts via a cell surface receptor
- coupling of hormone with a large molecule
- since compound cannot pass membrane, if a response occurs –> receptor is on cell surface - Limited proteolysis of intact cells would be expected to destroy the receptor and remove hormone response
The LH receptor is a G coupled receptor associated with Galphas type. What would be the effect of LH stimulation on cAMP levels in target cells? Why?
LH stimulation –> Galphas –> increases adenylate cyclase activity –> ATP used to increase cAMP levels
What would happen in cells were treated with forskolin?
Forskolin –> activates adenylate cyclase —> increased cAMP
A hormone X was shown to act via a G-coupled receptor associated with Galphaq type. What would depleting cellular calcium do to the hormone action? Why?
calcium is the primary effector and is responsible for activating calcium dependent PKC
- calmodulin binds to calcium and activates proteins
kinase phosphorylates protein –> biological response
Describe the genomic effects of cAMP
Activate protein kinase A –> phosphorylation of intracellular proteins –> immediate cell responses
- modification of metabolic pathways
- regulation of ion flows and muscle contraction
- effects of gene transcription by protein kinase A activation of the cAMP-responsive element binding protein (CREB), or modification of the structural proteins in chromatin
- Activated CREB minded to cAMP responsive elements in regulatory regions –> activate gene transcription
Describe experiments that could be performed to demonstrate that cAMP is involved in a particular hormone response
Can be determined if physiological levels of hormone increase cAMP in cells and cAMP production precedes the physiological effect
- treatment with exogenous cAMP or its analogues –> hormone response
- phosphodiesterase inhibitors –> decrease cAMP clearance and potentiate the response
- treatment of forskolin –> activates AC - binds directly to catalytic subunit to permanently activate it
Describe experiments to demonstrate the involvement of a particular G protein in a hormonal response
Measure activity of PLC, AC or does with IP3 and cAMP
Measure activation and activity of downstream factors (kinases and transcription factors) following activation/inhibition
Use specific blockers/activators:
- non-hydrolysable form of GTP or cholera toxin –> stimulates Galphas and AC –> cAMP levels increase
- pertussis toxin blocks Galphai –> no inhibition of AC –> cAMP levels do no decrease
Why are EGF and insulin receptors called tyrosine kinases?
Tyrosine kinase receptors:
- do not use a second messenger system to activate a separate protein kinase (but have a kinase domain as part of their structure)
- activated receptor phosphorylates tyrosine residues in its kinase domain and can then phosphorylate other proteins
Made up of 3 domains Extracellular domain: - ligand recognition and binding Transmembrane domain Intracellular/cytoplasmic domain: - auto-phosphorylation site - transmits regulatory signals and contains binding sites for signalling proteins
3 main classes
- Class I = EGF
- monomeric transmembrane protein with intracellular and extracellular domains on the same molecule - Class II = insulin
- heterotetrameric receptor - two alpha subunits and 2 beta subunits linked by disulphide bonds
Tyrosine kinase receptos mechanism of action
- hormone binding causes activation and dimerization of receptors
- have kinase as part of receptor –> kinase domains in monomers are auto-phosphorylated (kinase phosphorylates tyrosines and activates other target proteins)
Describe methods to determine if Ca dependent pathways are involved in any system
- increase intracellular calcium levels using calcium selection ionophores or liposomes loaded with calcium
- decrease intracellular calcium by chelating with EGTA, using calcium blockers or inorganic calcium antagonists
- use phorbol esters (which resemble DAG) to activate protein kinase C
- inhibit phospholipase C with U73122 to block action of hormone
Outline the mechanism of action of cytokine receptors
Receptors for GH, prolactin, erythropoietin, interferons, interleukins
- do not have intrinsic kinase activity
- receptor exists as inactive dimer when not bound to hormone
- binding hormone causes rotation of intracellular domains to align JAK tyrosine kinase bound to each receptor monomer
- JAK tyrosine kinase phosphorylates the receptor
- phosphotyrosine acts as docking site for intracellular signalling molecules
- STATs activate various genes
Outline mechanism of action of serine kinase receptors
- TGFB family mainly involved in control of cell proliferation and differentiation (inhibit, activin)
- binding of hormones –> formation of heteromer of receptor 1 and 2
- after hormone binds specific RII, the H-RII complex recruits RI (same RI can be recruited by different H-RII complexes)
- serines on RI are phosphorylated by RII
- activated receptor phosphorylates Smad proteins
- Smad dimerizes, translocates into nucleus and modulates gene transcription
Describe the role of MAP kinase in integrating the endocrine response to growth factors
- mitogen activated protein kinases - serine/threonine protein kinases which integrate various extracellular signals in response to growth factors (e.g. cytokines, stresses)
- MAPK-kinase and MAPK-kinase-kinase stimulated by the monomeric G protein RAS (which is activated by adapted molecules that bind to activated tyrosine receptors)
- cascade can also be activated by PKS and other G proteins
- MAPKs integrate cellular response to growth factors, cytokines, stressors
Explain the significance of finding that the monomeric G protein Ras is found in an active form in many cancers
Ras –> stimulates MAPK process –> responds to growth factors –> increased growth rate
