General Principles I

Objectives:- To know the 4 types of intercellular signalling (Contact Dependent, Paracrine, Synaptic, Endocrine).- To know that most signalling molecules are recognised by cell surface receptors.- To know that information is transferred by a change in state of the protein, with examples.- To know that signal integrations can alter cell state. (Context) - To know that the same signalling protein can induce different responses. (Context)- To be aware of the recurring theme of receptor activation and downstream signalling.- To know the effects of post-translational modifications on cellular signalling.- To know the structure and mechanism of protein kinases, protein phosphatase, monomeric GTPase,This lecture just gives a general overview of cell signalling.

What actually is Cell Signalling? This is the coordination of cellular activity between cells to respond to chemical and physical changes in their environment. Extracellular signalling molecules interact with cell surface receptors by docking. The receptor is activated by a conformational or structural change; thus binding activates intracellular signalling pathways within the cell that go on to target effector proteins. This process mediates cell behaviour such as: gene expression, protein production, catalytic states, cell cycle stage, ion channels, cytoskeleton modification etc.

5 types of intercellular signalling (Contact Dependent, Paracrine, Synaptic, Endocrine):(A) Contact DependentA membrane bound signalling molecule must physically meet between the membranes of two cells e.g. T-cell receptor triggering.(B) ParacrineA diffusible mediator is secreted that reaches a target cell in the local environment e.g. Growth factor signalling in tumour cells.(C) SynapticNeurotransmitter diffuses accross synaptic cleft and binds to receptor on post-synaptic neuron often triggering an action potential.(D) EndocrineChemical messenger hormones are secreted into the bloodstream that target a receptor on a cell e.g. Adrenergic receptors.(E) AutocrineWhen a chemical messenger or hormone is secreted from a cell to target receptors on itself e.g. cytokine interleukin-1 in monocytes.

Cell Surface Receptors:Most signal molecules are recognised by cell surface receptors. They change shape transferring the signal to the internal membrane structure. Intracellular Receptors:Some signal molecules are transported via carrier proteins across the cell membrane to target an intracellular receptor protein. A small hormonal hydrophobic signal molecule can be transported into the cellular cytoplasm where it can then bind to nueclear receptors e.g. 17-B-estradiol estrogen receptor (ER).

Signal Integration: Animal cells respond to multiple signal molecules such that signal integration dictates the behaviour of the cell to survive, grow and divide, differentiate or undergo apoptosis. It is worthy of note that the same signal molecule can induce different responses in different cells e.g. acetylcholine on the heart, salivary gland and skeletal muscle cell.1. Heart pacemaker cell: Aceylcholine binding leads to a decreased rate of action potential firing, thus a decreased heart rate.2. Salivary Gland Cell: Acetyl choline bindings causes the secretion of saliva.3. Skeletal Muscle Cell: Acetyl choline binding causes the contraction of muscle cells.Therefore the context of the cell type and cell cycle stage is important in cell signalling.

Different Signalling Strategies:1. Binding/Dissociation where activation is dependent on the binding or dissociation of an extracellular signal.2. Post-Translational Modification where on/off switch is dependent on a modification such phosphorylation by a kinase.3. Conformational Change where activation is brought about by a conformational change in receptor upon binding of signal molecule.4. Localization where transcription factors can achieve different changes in state coupled with long signalling cascades.Therefore there is information transfer across the plasma membrane (Receptor Activation) to trigger downstream signalling events within the cytosol to target effector proteins. These intracellular secondary messengers that target effector proteins can vary from:- Small water-soluble molecules: cAMP, Ca2+- Small lipid-soluble molecules: diaacylgrlycerol- Proteins that undergo post-translational modifications e.g. phosphorylation (kinase, phosphatase).- Proteins such as GTP binding proteins (GTPases).

Effects of Post-translational modifications:(a) Change in conformation altering activity where binding of intracellular signal alters conformation and activity of protein.(b) Promote protein binding where binding of IS induces binding of other units to protein.(C) Prevent protein binding where binding of IS prevents protein binding.(d) Changing subcellular localization where binding of IS to protein takes it to a specific part of cell e.g. nucleus.(e) Change in proteolytic stability where IS binding can break up or prevent protein degradation.

Protein Kinases: There are ~500 human protein kinases that have phosphorylating activity. These are subdivided into Ser/Thr kinases such as calmodulin-dependent protein kinase, protein kinase A and MAP kinase. Another subdivision is Tyr kinases such as the Src-family kinases and receptor tyrosine kinases.Structure & Mechanism: All have an N-lobe, C-lobe and an ATP substrate binding pocket. The mechanism revolves around a nucleophilic attack on a phosphate by the substrate (signalling proteins) where a phosphate moiety is transferred to the substrate. An activation loop is required to unblock the active site. All kinases have variations of this mechanism.Protein Phosphatases: Also ~140 protein phosphatases that remove phosphate moieties from substrates, These are divided into Ser/Thr phosphatases and Tyr phosphatases.Monomeric GTPases: GTPase is in its active state when GTP is bound, otherwise it is in its inactive GDP bound state. Ran-GEF can transfer a GDP for GTP to activate GDP bound GTPase. Ran-GAP hydrolyses GTP bound GTPase releasing a Pi to return to the inactive monomeric GTPase state. These states can influence cell signalling pathways.