Mechanisms of cellular interactionN

Cells do not exist in isolation. All cells are supported and regulated by mesenchymal-derived connective important tissue, an important element of which is the cell-secreted extracellular matrix consisting of polymeric proteins including collagens, elastic fibres and proteoglycans. Specialised cell surface molecules mediate cell-cell and cellular-extracelluar contacts to allow constant interactions between neighbouring cells, the extracellular matrix and soluble molecules.

ADHESION

The intercellular space is bridged by a series of transmembrane proteins connecting to cytoplasmic filamentous networks. These cell-cell junctional complexes have different properties according to the nature and the requirements of the cell. For example, tight junctions closely oppose neighbouring cells, providing a barrier of high electrical resistance, whereas gap junctions formed by the connexin family of proteins, function not as barriers but as intercellular passages for ions and small molecules.

cell matrix binding uses adhesion molecules as anchors between the cell surface and matrix molecules. The predominant extracellular matrix receptors on cells are integrins, which are transmembrane glycoproteins requiring cations for ligand binding. Precise adhesion molecules are often specific to the matrix component.

Numerous molecules are involved in particular processes, each mediating specific functions. As an example, adhesion molecules involved in the interactions between leucocytes and the endothelium are illustrated in Table 1.3.

EXTRACELLULAR SECRETION AND TRANSMEMBRANE PROTEINS

Proteins are generally secreted from the cell by exocytosis, a process whereby vesicles containing material destined for the cell exterior detach from the Golgi apparatus and are guided by microtubules to the plasma membrane, from which they may be discharged, often requiring an additional specific stimulus. Transmembrane proteins are retained within mem. hranes due to the presence of stretches of approximately 25 hydrophobic amino acid residues which anchor the protein in the non-polar interior, initially of the endoplasmic reticulum membrane, and subsequently the plasma membrane. The major groups of transmembrane proteins are illustrated . Once in the plasma membrane, proteins diffuse laterally and may cluster, either spontaneously or in response to specific stimuli.

OUTSIDE-IN COMMUNICATION

A cell needs to be able to internalise substances from its exterior--for example, for nutrient uptake, recycling or protein degradation. In addition, the cell needs to recognise changes in its environment and initiate suitable responses,which may be immediate (such as the reorganisation of existing protein structures leading to processes such as contraction or depolarisation), or which may depend on the synthesis of new proteins, initiated by transcription of a specific repertoire of genes.

Internalisation processes

The fastest way for small extracellular molecules to enter the cell is by passive diffusion along osmotic gradients, directly through the lipid bilayer if lipid-soluble, through an aqueous channel if highly charged or, in the case of water in the sell, itself,through aquaporin channel-forming proteins. Alternatively, carrier proteins may be required to facilitate passive transport by conformational change, randomly opening first to the exterior and then to the interior of the cell, allowing different ions to diffuse along their concentration gradients. Ina limited number of cases, transport against concentration gradients is possible by the synchronous transport of another molecule down its concentration gradient.

Endocytosis and associated mechanisms are used for internalising larger particles (< 0.2 um in diameter). These mechanisms, include the invagination of simple plasma membrane pits (caveolae), or engulfment, particularly of larger particles, by pseudopodia which extend from the plasma membrane due to the polymerisation of filamentous F-actin. Such phagocytosis, which is particularly observed in macrophages and neutrophils, may invaginate up to 50% of the membrane surface area. On a smaller scale, membrane ruffling (also an F-actin-dependent process) may lead to occasional fusion and the invagination of small particles in a process known as micropinocytosis. Vesicles formed by these processes fuse rapidly with endosomes and subsequently lysosomes. However, not all noxious substances are subsequently degraded-for example, mycobacteria modify the endosome/lysosome system and remain viable in the altered vacuoles.

Receptor-regulated cellular entry

The processes described above would not allow for specific regulation of membrane transfer to permit the transient flux of ions such as K+, Na,+ ,Ca++ or Cl- at designated times to alter intracellular environments, or initiate membrane depolarisation. Many ion channels open only after a specific signal has been received. This may be from within the cell, such as an alteration in voltage, intracellular volume Ca++ concentrations, or from the extracellular environment, often mediated by G-protein coupled receptors which are examples of type 3 transmembrane proteins . Such events may be relatively simple receptor-mediated events. For example, acetylcholine induces membrane depolarisation by binding to the nicotinic cholinergic receptor which undergoes a conformational change to permit Na influx. Other ion channels depend on signals generated by more complex intracellular pathways (see below).

Analogous receptor-mediated mechanisms operate to allow the internalisation of moderate-sized particles using clathrincoated pits. The low-density lipoprotein (LDL) receptor operates in this way; hereditary defects lead to a failure in uptake of cholesterol from the plasma, and the consequences of familial hypercholesterolaemia.

Cells use energy to set up concentration gradients which can be subsequently exploited by the rapid and regulated opening of specific ion channels. For example, the cytosolic Ca++ concentration is maintained at 10 to the power-7 M, compared to an extracellular concentration in the order of 10 M by two calcium transporter pumps, one driven by ATPase, the other by the coupled transport of sodium ions in the opposite direction. Similarly, ATP is hydrolysed to provide the energy for the Na*/K* ATPase to transport sodium ions into and potassium ions out of the cell, maintaining osmotic homeostasis.

Signal transduction

As noted with acetylcholine, a molecule does not have to enter a cell in order to alter the cellular environment. Instead, molecules may be recognised at the cell surface by a specific receptor. Binding of the extracellular ligand activates the receptor, which then initiates events which may alter the cellular structure directly (e.g. by depolarisation).

In more complex reactions, the signal is propagated downstream via a number of intermediate signalling moieties. Ligand binding generally leads to receptor activation, and the activated receptor then stimulates a series of other membrane-bound or cytoplasmic signalling molecules, which in the final stages may translocate to the nucleus to alter nuclear transcription, or interact with other cytoplasmic and membrane components. Key features of such cascades are to allow for substantial amplification of the initial singal, and to permit a coordinated cellular response,since more than one series of downstream events may be triggered. This results in the components of these pathway being numerous and potentially confusing. Several different types of signalling cascade are illustrated in a simplified form, showing how the cell responds to major families of ligands-for example, hormones, inflammatory cytokines and growth factors.

Second messengers

Phospholipase cascades. Many receptors trigger the hydrolysis of phospholipids within the plasma membrane to generate a series of specific second messengers. For example, phospholipase C (PLC) cleaves phosphatidylinositol 4,5-bisphosphate (PIP) into two short-lived messengers, diacylglycerol (DAG) and inositol 1,4,5trisphosphate (IP3). DAG activates protein kinase C and initiates further downstream activation of signalling pur proteins. IP3: causes the rapid release of calcium from intracellular stores by opening calcium channels in the endo- plasmic reticulum (and sarcoplasmic reticulum in smooth muscle cells). Calcium is a particularly good messenger for rapid release since, as mentioned above, extrusion of calcium maintains tightly controlled low intracellular concentrations allowing rapid re-uptake of the molecule for signalling purposes. Intracellular calcium ions can then interact with a number of proteins which contain calcium recognition motits, such as a helix-loop-helix motif known as an EF hand, initiating further downstream events. Examples include the activation of protein kinase C (DAG increases its affinity for calcium) and calmodulin which, when activated by calcium binding, stimulates mutifunctional kinases such as calmodulin-dependent protein kinase II which regulates neurotransmitter release and ionic permeability Receptor mediated activation of other phospholipases, including phospholipase D and phospholipase A2, operates by similar principles, generating the second messengers DAG and arachidonic acid respectively.

Cyclic nucleotide cascades. Numerous signalling pathways stimulate transmembrane adenylyl cyclase to convert ATP to adenosine 3'.5'-cyclic monophosphate (CAMP), O guanylyl cyclase to convert guanosine triphosphate ( GTP) to guanosine 3',5'-cyclic monophosphate (CGMP). These pathways include hormonal signalling via G-protein coupled receptors (e.g. B2-adrenoceptor causing accumulation of cAMP), and nitric oxide which stimulates a cytosolic guanylyl cyclase to produce cGMP, initiating a cascade which in appropriate cell types culminates in vasodilatation. Responses to these cyclic nucleotides often depend upon activation of downstream enzymes. For example, cAMP activates the enzyme protein kinase A, which can open ion channels directly, alter cellular metabolic pathways, or alter nuclear transcription by binding to a CAMP-response element binding protein (CREB), which binds to calcium/cAMP-response elements in promoters of genes such as c-fos.

Phosphorylation cascades

Many signalling cascades involve the transfer of phosphate groups to molecules to render the phosphorylated product enzymatically active as a kinase, enabling the subsequent transfer of phosphate groups in further catalytic reactions downstream. Such cascades often commence with the activation of a receptor following ligand binding,by phosphorylating tyrosine or serine and threonine residues on receptor subtype. The progress of the cascade usually involves serial phosphorylation of cytoplasmic proteins.

Recognition domains. Intracellular signalling proteins often have motifs to allow them to recognise and interact with activated (phosphorylated) molecules in signalling cascades. Several families of recognition motifs are recognised and designated by their homologies to key signalling molecules, e.g. Src homology (SH2 and SH3) and plextrin homology (PH) domains. Most signalling proteins have at least two of these motifs, which effectively create 'docking sites' to allow correct orientation of a series of proteins.

Phosphorylation switches. The phosphate groups involved in the immediate phosphorylation of these receptors are often derived from the y-P of ATP, whereas the phosphate groups used to phosphorylate cytoplasmic signalling proteins are generally derived from GTP, bound to a series of guanine-nucleotide binding proteins (G-proteins). The cytoplasmic provision of GTP is regulated by proteins of the Ras family which carry guanosine diphosphate (GDP) that is converted to GTP by activated upstream signalling molecules. The deactivation of Ras proteins is accelerated by GTPase-activating (GAP) proteins which may be inhibited by growth factor-receptor interactions. These series of effector molecules are conserved signalling molecules from yeast to humans. Aberrant activation of Ras-GTP molecules provides a continuous signal for cell replication in many cancers. Other examples include the Rab family of Ras-like GTPases which provide the energy for vesicle transport , and neurofibromin, a Rasregulating GAP, the gene for which is mutated in neurofibromatosis.

Mechanisms of retaining a specific cellular response

At first sight it may appear that despite specificity at the level of the ligand-receptor interaction, the signalling cascades converge on common pathways and will be able to generate only a limited number of cellular responses. Specificity therefore relies on the repertoire of receptors and signalling proteins expressed by individual cells, the precise magnitude and timing of the signalling events, and the functional responses available to a particular cell type.

Mechanisms of desensitisation and tolerance

Most signalling pathways are controlled by activated components with short half-lives, and negative feedback loops. In addition, a number of them can become resistant to continued ligand stimulation, a process critical in the repeated administration of drugs. Mechanisms inducing such downregulation include receptor desensitisation, up-regulation of downstream signalling moieties and up-regulation of transporters to displace intracellular molecules from sites of activity .