Cell biology lecture 16

More signalling through cell surface receptors

3 types of cell surface receptors

G-protein activation of downstream signalling pathways​

2 key proteins activated by the trimeric G proteins are:

• Adenylyl cyclase​

• Phospholipase C​

These enzymes pass information down the signalling pathway by producing 2nd MESSENGERS​: See picture

Learning objectives

• IP3 and DAG as secondary messengers​

• Protein kinase C​

• Calcium as a secondary messenger​

• What are Enzyme linked receptors?​

• Receptor tyrosine kinases (RTKs)​

• The Ras-MAPK pathway and cancer​

Activation of the G-protein linked receptor​

Results in dissociation of the trimeric G-protein

Transduction of G protein signals

Phospholipase action

Cleaves phosphoinositol 4,5-bisphosphate (PIP2) to produce IP3 and DAG​

IP3 and DAG action

Ca2+ as a second messenger

• Small changes in Ca2+ are easily detected, because cytosolic Ca2+ levels are maintained at a low level (~10-7M), compared to extracellular Ca2+ (~10-3M) ​

• Ca2+ can bind tightly to proteins inducing conformational change​

Calmodulin

Mediator of many calcium dependent effects

• Each calmodulin molecule binds 4 Ca2+ ions​

• The resulting conformational change allows the calmodulin/Ca2+ complex to wrap around and activate target proteins​

Enzyme linked receptors

• Single-span transmembrane proteins​

• Cytosolic domain has intrinsic enzymatic activity or is associated with an enzyme​

Receptor Tyrosine kinases (RTK)​

• most common type of enzyme linked receptors​

• The RTK family includes Insulin receptor and many growth factor receptors​

• Growth factor receptors control cell proliferation​

• Cancer is frequently associated with problems in growth factor signalling ​

What does the cytoplasmic/cytosolic domain or receptor tyrosine kinases have?

Intrinsic kinase activity

• Binding of the ligand to growth factor receptors leads to cross-linking of two receptor chains​

• Oligomerisation of the receptor chains allows cross-phosphorylation (autophosphorylation)​

• Insulin receptors are tetramers; ligand binding causes realignment of the polypeptide chains activating cross-phosphorylation​

what do phosprylated tyrosine residues achieve?

Provide docking sites for other signalling proteins

The RAS Signalling Pathway​

• Ras is the main signal-transducer protein for growth factors e.g. Fibroblast growth factor (FGF), epidermal growth factor (EGF)​

​

• Ras is a small MONOMERIC G-protein (very small)​

Trimeric protein

• Like G protein coupled receptor (larger protein)

• Bind directly to receptor ​

• Receptor activates GDP release​

• GTP hydrolysis by intrinsic GTPase activity alone​

Monomeric protein

• Like RAS

• Not directly linked to receptor​

• GDP release activated by GEF (guanine nucleotide exchange factor)​

• Weak intrinsic GTPase activity – needs GAP (GTPase activating protein) to drive GTP hydrolysis.​

RTK mediated activation of the RAS-MAPKpathway​

• RAS and GEF do not bind directly to the RTK​

• Binding is mediated by an adapter protein, Grb-2

What does Ras activate?

A downstream phosphorylation cascade

Ras, Raf, Mek, Erk

Amplification steps of the Ras-MAPK pathway:​

The RAS Signalling Pathway and cancer​

• RAS is a proto-oncogene (in cell growth)​

• RAS mutations are found in 20-30% of human cancers (~80% pancreatic)​

• Most common RAS mutations reduce GTP hydrolysis activity​

• GTP stays bound longer and the signalling pathway is continuously switched on​

• Leads to cell proliferation, even in the absence of growth factors such as EGF

• RAS becomes an oncogene and causes cancer​

Ras pathway

Inhibitors targeting The RAS Signalling Pathway​

Don’t need to know specific drugs