Membranes & Cell Signalling

Department of Bioscience

Lecture Notes: Membrane Signalling Complexes

Background reading:
chapter 20, Lodish 4th ed. http://www.whfreeman.com/lodish/
Lipid Rafts Lecture notes

Additional reading:
Pawson, T, Gish, GD & Nash, P (2001) SH2 domains, interaction modules and cellular wiring. Trends Cell Biology 11, 504-511.
The random collision model

predicts the lateral movement of membrane receptors and membrane-associated proteins freely through the lipid bilayer
interaction of active receptors and other signalling enzymes occurs through random collisions between the proteins with specificity determined by the sites of protein-protein interactions
the diffusion rate of each molecule within the lipid bilayer influences speed of signalling

Evidence for a more highly organised state

limited mobility of receptors and G proteins does not fit into random collision model

in particular, the bg subunits of heterotrimeric G proteins show a very restricted mobility

receptors do not have fee access to all G proteins with which they are capable of coupling
different receptors that are capable of coupling to the same G proteins do not seem to share the same pool in intact cells

Membrane receptors and associated signalling molecules can be highly organised and compartmentalised into membrane signalling complexes in order to form biochemical pathways or networks

Confers specificity, precision and speed

Key features of membrane signalling complexes:

1. Immobilisation and clustering of receptors and signalling molecules

2. Correct targeting to specific subcellular sites

3. Confers ability to funnel ionic and conformational signals to appropriate cytoplasmic effectors

4. Association with specific modulatory proteins such as kinases, phosphatases and other regulatory proteins

5. Signal amplification

through adapter proteins which provide additional docking sites for other modular signalling proteins
through reiteration of specific protein-protein interaction motifs present within an individual docking protein

Compartmentalisation mechanism can involve interactions between receptor-signalling complexes and membrane or cytoskeletal proteins via adapter/scaffolding proteins. Adapter/scaffolding proteins have no obvious catalytic function, and act to assemble other components into the signalling complex

Fig. 20-5c, Lodish, 4th ed.

Protein scaffolds have two related functions

1. maintain the specificity of the signalling pathway (either isolating or stabilising interactions between the kinases of a single cascade)
2. catalyse the activation of the pathway components (by holding the kinases in a manner that directly enhances their mutual interactions)

Anchoring scaffolds

function to co-localise a group of molecules that participate in the same signalling process to a specific area of the cell, enhancing the efficiency of signal propagation

Catalytic scaffolds

see MAP kinase signalling (Figs. 20-31, 20-33 in Lodish)

Signalling complexes give a temporal and spatial quality to signals. Co-localisation of components of a signalling complex can occur statically or dynamically - both of these often occur within the same cell but for different purposes.

Assembly of signalling proteins into biochemical pathways or networks

Activation of dormant enzymes already positioned close to their substrates

In the static model, the signalling complex components are held together (quite often in a dormant state) by scaffolding proteins before the signal arrives. Enables second messengers and a small number of diffusible proteins to transmit information spatially across the cell

time dependent
concentration dependent

Examples of second-messenger based spatial control mechanisms include

Cell-wide Ca²⁺ spikes
Self-propagating Ca²⁺ waves

The advantage is that cells can rapidly transfer information within these complexes and use the relatively fast diffusion of second messengers to activate cellular responses with minimal delay.

Active recruitment of signalling molecules into multi-protein networks

In the dynamic model, signalling proteins are reversibly bound to adaptors, the cytoskeleton or membranes. Between being localised in one of these transient complexes, signalling molecules readily diffuse within the cytosol or membranes. The transient translocation of signalling proteins to and from specific sites and the assembly of signalling complexes are essential for generating efficiency and specificity. Examples of active recruitment of signalling molecules into a multi-protein network include

The advantages of having protein translocation steps in a signalling pathway?

can generate more diverse cellular responses by introducing additional steps for assembling signalling complexes

enables a high degree of regulation of these processes

suppress subthreshold inputs by requiring stimuli to be sufficiently long or strong for translocation to occur
delay a signalling step

through slow diffusion and on-rates in assembling the complex

prolong a signalling step

through slow off-rates in disassembly of the complex

decrease or increase rapidly the number of available signalling proteins

by binding to or release from other, more-abundant sites

Many of the signalling protein components as well as the receptor itself consist of modular domains which determine protein-protein interactions necessary for the recruitment of particular proteins into signalling complexes. (These are called modular domains because they are usually a small stretch of the polypeptide sequence which folds into a discrete domain)

SH2, SH3, PTB, PH domains and others such as WW domains (small modules of 35-40 residues which bind proline rich motifs )
PDZ domains

Individual protein components can consist of different combinations of modular domains. Furthermore, many docking proteins have an N-terminal PH domain or myristilation site (targeting them to the membrane) in combination with other protein binding domains and/or catalytic domains

Adapted from Pawson, T, Gish, GD & Nash, P (2001) Trends Cell Biology 11, 504-511.
Protein modular domains determine specificity of protein-protein interactions

SH2 (Src homology) domains

Phosphotyrosine binding domain
tertiary structure of domain (depends on primary amino acid sequence) recognises specific binding motif
Recognition is determined by amino acid residues C-terminal to the phospho-tyr of the binding partner

PTB domains

Phosphotyrosine binding domain
Structurally differs from SH2 domains
Recognition is determined by amino acid residues N-terminal to the phospho-tyr and highly dependent on its 3D conformation

SH3 domains

Recognises proline rich sequences which commonly contain the PXXP motif, thus forming a lefthanded polyproline type II helix
Regulated by Ser/Thr phosphorylation at nearby sites
Thought to form bridges between signalling molecules and with the cytoskeleton

PH domains

Pleckstrin homology domains
Overall sequence similarity very low between different PH domains
PH domain proteins tend to be membrane associated and/or in the inositol phosphate signalling pathway

Principal binding partners: phosphoinositides, G protein bg subunits

May be different functional subfamilies

Mediate protein-protein interactions
Mediate protein-lipid interactions

PDZ domains

~90 amino acids
Bind to consensus carboxyl-terminal motif of target protein (-[Ser/Thr]-X-Val)

specificity conferred by the -2 and -4 positions relative to the carboxy-terminus

Play an important role in the spatial organisation of voltage- and ligand-gated ion channels at synapses
proteins with up to 7 PDZ domains have been characterised

Active recruitment - Receptor Tyrosine Kinases

RTKs can amplify their signal by using adapter proteins that provide additional docking sites for modular signalling proteins
the assembly of complexes of signalling proteins around an activated cell-surface receptor is directed by protein modular domains present within the individual molecules
commonly, specific protein-protein interaction motifs may be reiterated several times within an individual docking protein, allowing amplification of specific signalling pathways

Protein components recruited into signalling complexes

Enzymes that control phospholipid metabolism
Ras-like GTPases
Protein kinases and phosphatases
Transcription factors (such as STATS - see cytokine receptor signalling)
Polypeptides that regulate cytoskeletal architecture and cell adhesion

Activation of multiple signalling pathways Different receptor phospho-tyr motifs in an individual receptor can recruit different signalling components and trigger multiple signal transduction cascades.

Activation of the PDGF receptor:

1. Binding of the SH2/SH3 domain containing adapter protein Grb2 to the active receptor

recruits GTP exchange factor Sos (son of sevenless) which contains proline rich sequences. Sos in turn catalyses the exchange of GDP for GTP for the small G protein Ras. Activated Ras leads to activation of several targets, in particular the Raf/Map Kinase pathway.

2. Binding of PLC-g through its SH2 domain to the active receptor

leads to its phosphorylation and activation. PLC-g catalyses the hydrolysis of the membrane lipid PIP2 into DAG (an activator of protein kinase C) and IP3 (which mobilises calcium from intracellular stores).

3. Binding of PI3-kinase to the active receptor through its regulatory (p85) subunit

leads to its phosphorylation and activation of the catalytic subunit (p110). The regulatory subunit is an adapter protein and contains both SH2 and SH3 domains as well as proline rich sequences. PI3-kinase is a lipid kinase but also has ser/thr kinase activity. Evidence suggests that PI3-kinase also can interact with and activate Ras.

Static complexes - Important adapter/anchoring proteins involved in signalling

PDZ domain proteins
Homer (metabotropic glutamate receptor binding proteins)
14-3-3 proteins (dimers - linking together signalling components by phospho-Ser binding, also known as kinase binding proteins)
AKAPs (PKA and associated proteins)
PKC binding proteins: substate binding proteins, RACKs, PICKs

PDZ domain protein families

Postsynaptic density proteins (PSD-95/SAP90)
CASK
GRIP/ABP
S-SCAM
Mint1
PICK1

PDZ interactions can co-ordinate the localisation and clustering of receptors and channels, and provide a bridge to the cytoskeleton or intracellular signalling pathways through multiple types of protein:protein interactions
Multiple PDZ domains can induce channel aggregation through binding to several channel proteins via particular subunits

PDZ domain proteins can oligomerise through N-terminal disulfide bonds, and enhance aggregation
Varying PDZ domains bind different motifs, thus complexes containing multiple proteins can form
Interactions between PDZ domains can direct homotypic or heterotypic interactions with other PDZ domain proteins

Signalling complex maintained by PDZ interactions necessary for phototransduction in Drosophila eye

Anchoring PDZ domain protein InaD (Inactivation No-After Potential), which contains five PDZ domains
Associates through distinct PDZ domains with a calcium channel (Trp), phospholipase C-b (target of rhodopsin-activated Gqa), protein kinase C, calmodulin, and InaC
Signalling complex allows efficient activation of TRP channel by PLC-b in response to light stimulation and subsequent deactivation of TRP channel by PKC phosphorylation

Scaffolding for serine/threonine kinases - AKAPs (A Kinase Anchoring Proteins)

Multivalent anchoring proteins which bind regulatory subunits of PKA

25 AKAPs characterised

different gene families
numerous splice variants and isoforms

Some have the ability to maintain signalling scaffolds by simultaneously binding to other protein kinases and phosphatases (eg PKC, calcineurin)

Maintains enzymes close to their substrate(s)

Some are found to associate with ion channels (eg. glutamate receptors) as well as receptor proteins (eg. b2-adrenergic receptor)

regulating the phosphorylation state and activity of transmembrane proteins

Each contain specialised targeting regions determine localisation to specific subcellular compartments

Polybasic regions
Myristoylation modification targets protein to membranes
Direct protein-protein interactions to structural elements
Direct protein-lipid interactions to membranes

Regulation of anchored enzyme activity is emerging as an important property of AKAP complexes

Enzymes are inhibited (inactive) when bound to AKAP scaffold - catalytic subunit of PKA, PKC
Upregulate activity of anchored phosphatases, such as PP1 (Yotaio complex)

PKA anchoring ensures that kinase is exposed to localised changes in cAMP (through concerted actions of adenylyl cyclase and phosphodiesterases) and is positioned near ion channels or other substrates whose activity it modulates

Kinase signalling cascades - MAP kinase pathways (see also Kinases & Phosphatases Lecture notes)

A highly conserved cascade of protein kinases participate in sequential fashion to activate MAP kinase in response to various extracellular signals. Different scaffolding proteins may be involved in anchoring these kinases

Proteins thought to be involved in the Ras/Raf activation of MAP kinase

Ksr - contains binding sites for Raf, 14-3-3, MEK and MAP kinase
14-3-3 proteins - binds Raf

Fig. 20-31, Lodish 4th ed. Example: The MAP kinase cascade of Saccharomyces cerevisiae pheromone response pathway is organised on the Ste5 protein, which binds each of the kinases of the cascade prior to signalling. This cascade is linked to GPCRs for two secreted peptide pheromones, the a and a factors, which control mating between haploid yeast cells of the opposite mating type, a or a. Activation requires the Gbg subunits, rather than the a subunits.
Gbg : Ste20 (Ser/Thr kinase) : Ste11 (Ser/Thr kinase analogous to Raf) : Ste7 (dual specificity MEK) : Fus3 (MAP kinase) which phosphorylates Ste12 (transcription factor involved in mating response)

Lecture notes updated 16/2/2002

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