RTK structure, activation, and intracellular signalling coupling

GPCRs

-        Transmembrane proteins with their ligand binding domains on the outer surface of the PM

-        Cytosolic domain associates with a trimeric G protein

-        Seven transmembrane segments

Enzyme Coupled receptors

-        Transmembrane proteins with their ligand binding domains on the outer surface of the PM

-        Cytosolic domain either has intrinsic enzyme activity or associates directly with an enzyme

-        Each subunit of an enzyme-coupled receptor typically has only one transmembrane segment

-        Receptor tyrosine kinases are a class of enzyme coupled receptors and is the most common

-        Many extracellular signal proteins act through RTKs

There are about 60 human RTKs which can be classified into about 20 structural subfamilies

Activation of RTKs

1.      Binding of signal protein to ligand-binding domain on EC side of receptor

2.      Activates tyrosine kinase domain on cytosolic side

3.      Phosphorylation of tyrosine side chains on cytosolic part of receptor

4.      Creates phosphotyrosine docking sites for various intracellular signalling proteins

How does the binding activate the kinase domain on the other side of the plasma membrane

For GPCRs the ligand binding causes a conformation change

For most RTKs ligand binding causes the receptors to dimerise bringing the two cytoplasmic kinase domains together which promotes their activation

-        RTK receptor activation by trans-autophosphorylation such as insulin receptors

o   Leads to dimerization of the RTK which leads to the phosphorylation of each other

-        RTKs receptor activation through conformational change, but very change to that in GCPRs, a mild conformation change. Used in the EGF receptor

o   Forms an asymmetric dimer as one is the receiver and the other is the activator

Phosphorylated tyrosine’s on RTFs serve as docking sites for intracellular proteins

-        Many intracellular proteins can bind to activated RTKs

-        Specific intracellular proteins will bind to specific phosphorylation sites on activated receptor

Once bound to activated RTK a signalling protein can

-        Become phosphorylated on tyrosine and become activated

-        Binding alone may be sufficient to activate signalling proteins by

o   Inducing a conformational change

o   Bringing it near the protein that is next in the signalling pathway

Different RTKs bind different combinations of signalling proteins so activate different pathways and responses

Some RTKs use additional docking proteins to enlarge the signalling complex, through scaffolding proteins, like insulin receptor recruits’ insulin receptor substrate 1 (IRS1)

Signalling proteins downstream of TRKs are diverse but usually share highly conserved phosphotyrosine-binding domains

-        SH2 domains, Src homology region

-        PTB domain, phosphotyrosine-binding

Many signalling proteins also contain other interaction domains

-        Grb2 is an adaptor protein containing SH2 domains and 2 SH3 domain it binds to IRS1 using SH2 domain and with its SH3 domain can bind to other proteins at proline-rich motifs

-        Allows enlarging of signalling complex

RTKs, Ras and Ras signalling

The GTPase Ras mediates signalling by most RTKs

-        Small monomeric GPCRs also activate Ras

Ras is a superfamily, RTK can lead to the activation of members of the Ras and Rho subfamily

Like other GTP-binding proteins, Ras functions as a molecular switch

-        Activated when bound to GTP.

-        Inactivated when bound to GDP

-        Ras GTPase activating proteins (Ras-GAPs) cause the phosphorylation of GTP to GDP inactivating

-        Ras guanine nucleotide exchange factors (Ras-GEFs) swap GDP for GTP activating

RTKs can activate Ras by

-        Activating a Ras-GEF, this is used in most cases

-        Inhibiting a Ras-GAP

 The activation of Ras is usually short lived

-        Tyrosine-specific protein phosphatases quickly shut off activated RTKs

-        Ras GAPs inactivate Ras

Ras activates a MAP kinase signalling module

Activated Ras interacts with several families of effector proteins

30% of all humans contain mutations in Ras

-        Very important oncogene

-        Can be constantly activated which changes gene expression to promote uncontrolled cell proliferation

-        Ras mutations, KRAS (85%); NRAS (15%); HRAS (>1%)

o   All compromise GTPase activity of RAS

-        GAP deletions, RAS can be activated in tumour by loss of GAPs

o   Most significant example is the loss of neurofibromin which is encoded by the NF1 gene

-        Growth-factor-receptor-activation, RAS signalling pathways are also commonly activated in tumours in which growth-factor-RTKs have been overexpressed or mutated

-        Mutation of amplification of RAS effectors, like BRAF is frequently activated by mutation in human tumours

-        EGRF and ERBB2 (also known as HER2/neu) are frequently overexpressed in many types of cancer

o   Monoclonal antibodies (Herceptin) bind to receptor and prevent the ligand from binding

RTKs and PI3K signalling

RTKs can bind and activate PI 3-Kinase

One of the proteins that binds the intracellular tail of RTKs in the plasma-membrane-bound enzyme phosphoinositide 3-kinase (PI 3-Kinase)

-        Phosphorylates inositol phospholipids rather than proteins

-        Both RTKs and GPCRs can activate it

-        Plays a central role in promoting both cell survival and growth

The generation of phosphoinositide docking sites by PI3-K

-        Can activate phospholipase C-γ as well

PIP3 can serve as a docking site for various intracellular signalling proteins which assemble into signalling complexes which relay into the cell. PIP3 remains in the PM until specific phosphoinositide phosphates dephosphorylate it, the most prominent being PTEN. It is not cleaved into PI2

Intracellular signalling proteins bind to PIP3 via a specific interaction domain such as a pleckstrin homology (PH) domain

-        PH domains function as protein-protein interaction domains

-        Only a small set of them bind PIP3

-        At least some of these also recognise a specific membrane-bound protein as well as PIP3

o   This greatly increases the specificity of binding

-        PH domains occur in about 200 human proteins 

o   Ras GEF sos which is the Ras specific GEF

o   Serine/threonine protein kinase Akt (PKB) in insulin signalling

§  Activated Akt affects multiple cellular targets which increase metabolism, growth synthetic process and proliferation and suppresses apoptosis

RTKs and GPCRs activate overlapping signalling pathways

-        Even when they activate different pathways the different pathways can converge on the same target proteins