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Cell physiology II
Basic science
Cell physiology II
the resultant effect. Synaptic transmission can be considered a form of paracrine transmission, with the chemical signal termed a ‘neurotransmitter’. Long-distance intercellular signalling utilizes the bloodstream to transport the chemical signal (hormone) from the generating (endocrine) tissue to the target cell. Hormones are examples of ligands that bind to surface and intracellular receptors. Peptide hormones (e.g. antidiuretic hormone) bind to extracellular receptors; steroid hormones (e.g. aldosterone) are lipophilic and readily cross the cell membrane to bind to intracellular receptors.
Christopher Fry
The speed of response varies greatly in the different modes of signalling. • For long-distance signalling, neurotransmission is the most rapid (seconds) because electrical signals convey information once the neurotransmitter has crossed the synaptic cleft. • Hormones that bind to surface receptors have a reasonable response time (minutes) because they initiate cellular responses relatively rapidly once they have reached the target cell. • Hormones that bind to intracellular receptors have the slowest response (hours) because they modulate transcription of deoxyribonucleic acid.
Abstract For multicellular organisms, specialization of function has evolved to enable different tissues to carry out specific tasks crucial to the survival of the entire organism. Communication between different cells is vital to coordinate the activity of these separate functions. Individual cells within a tissue must be oriented appropriately to fulfil organ function and structural integrity of the tissue must be maintained. This contribution considers how such group activity is achieved.
Keywords cell signalling; calcium; receptors; autocrine; intracellular; cell- surface; enzymes; G-proteins; cell junctions; tight junctions; anchoring junctions; gap junctions; extracellular; proteoglycans; collagens; integrins
Autocrine signalling: allied to intercellular signalling is the ability of some cells to influence their own activity through the release of chemical signalling molecules. Such autocrine signalling may be a positive feedback process, whereby the release of a particular ligand may accelerate a particular cell function, including a greater release of the ligand.
Cell signalling Basic concepts of cell signalling Type of signal: cells can communicate via chemical mediators or by electrical signals (the latter are considered below with respect to gap junctions). Chemical – in general, the target cell responds to chemical signals through a receptor that often has a very high affinity for a particular signalling molecule. A few signalling molecules can cross the cell membrane easily and directly regulate intracellular reactions. An example is the volatile gas nitric oxide, produced by the action of nitric oxide synthase, the activity of which is modulated by Ca2+ released in response to agents such as acetylcholine. Nitric oxide activates guanylyl cyclase to produce cyclic guanosine monophosphate to generate cellular responses such as relaxation of smooth muscles, as in vascular tissue or the corpus cavernosum.
Differential response of ligands: a particular ligand may generate different responses in different cells. This is because receptors to a particular ligand can be of several types and subtypes that elicit different intracellular responses; also, a particular subtype may initiate different responses in various cells. An example is the ligand acetylcholine, which can bind to nicotinic or muscarinic receptors and can mediate a variety of responses (Table 1). Intracellular receptors Intracellular receptors are a group of transcription factors that depend upon interaction with ligands: they include steroid hormones (including the mineralocorticoids, glucocorticoids and sex hormones), vitamin D and thyroid hormone. Figure 1a shows the general scheme: a lipophilic signalling agent diffuses into the cell, binds to receptors and initiates a series of steps to generate products that mediate the response of the hormone. Different cell types may have the same steroid receptor, but generate dissimilar
Receptors are proteins on the cell surface that initiate cellular reactions when a chemical (ligand) binds; or they are intracellular receptors that require the ligand to cross the cell membrane. Some signalling molecules act locally and must be rapidly removed or immobilized so that they do not to travel too far. An example of such a paracrine mediator is histamine, released from mast cells and basophils when sensitized by antibodies to generate local effects such as vasodilation or bronchoconstriction. Such local action is not sufficient in larger multicellular organisms, and target cells such as nerves have developed long axons to propagate
Effects of acetylcholine • Nervous transmission at autonomic ganglia • Excitation of skeletal muscle at the motor endplate • Contraction of various smooth muscles • Slowing of pacemaker rate at the sinoatrial node • Stimulation of secretion by salivary glands
Christopher Fry DSc is the Professor of Cell Physiology at Royal Free Hospital, London, UK. Conflicts of interest: none declared.
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Table 1
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© 2007 Elsevier Ltd. All rights reserved.
Basic science
Signalling by intracellular receptors and surface enzyme receptors
a
Steroid
b GTP Activated Ras
MAPKKK (Raf)
Ligand Primary response
Intracellular receptor
GNRP
MAPKK (MEK 1/2)
Receptor tyrosine kinase
DNA
MAPK (ERK 1/2) GAP GDP
Secondary response products
a Signalling by lipophilic agents, typified by a steroid hormone. The steroid diffuses into the cell and binds to an intracellular receptor. This complex facilitates transcription to generate several primary products. These in turn initiate transcription of genes for a number of secondary products that mediate the cellular response, and limit the production of further primary products. The same ligand and intracellular receptor regulate the transcription of different genes in different cells, enabling specificity of action. DNA: Deoxyribonucleic acid.
Inactive Ras
Target proteins in nucleus
Target proteins in cytoplasm
Cell membrane b Steps in the mitogen-activated pathway. A receptor tyrosine kinase (RTK) binds a ligand (e.g. growth factor) and initiates a series of intracellular steps that starts with activation of a membrane-bound monomeric GTPase, Ras. Ras is active when it binds guanosine triphosphate (GTP) and inactive when it binds guanosine diphosphate (GDP); the switch between the two states can be governed by GTPase-activating proteins (GAPs) and guanine nucleotide release proteins (GNRPs). RTK can activate Ras by activating a GNRP or by inactivating a GAP. Once activated, Ras initiates a further series of phosphorylations, culminating in activation of a mitogen-activated protein kinase (MAPK), which can activate nuclear and cytoplasmic proteins to initiate cellular events. Some MAPKs are known, including the extracellular signal-regulated kinases (ERKs). The intermediate steps in the MAP kinase pathways involve successive kinases; MAPK kinases (e.g. MEKs – MAP-ERK kinases); and MAPK kinase-kinases (e.g. Raf).
Figure 1
and tyrosine- associated receptor kinases. Ligands include many growth and differentiation factors, such as: • epidermal and nerve growth factors • vascular endothelial growth factor • platelet-derived growth factor. Upon activation, the receptors transfer a phosphate group from ATP to specific tyrosine moieties on the intracellular domain of the receptor or closely associated proteins. Many cytoplasmic proteins bind to the phosphorylated tyrosines to initiate cellular responses, including the Ras proteins, which stimulate proliferation and differentiation of cells. Figure 1b shows that a cascade of reactions involves mitogen-activated protein kinases and activation of regulatory gene proteins. Other enzyme-linked receptor pathways include the serine/ threonine protein kinases, which bind transforming growth factor-β proteins that initiate similar intracellular pathways through the mediation of Smad proteins. The receptor guanylyl cyclases, which bind atrial natriuretic peptides, generate cyclic guanosine monophosphate to mediate intracellular responses.
cellular responses. This is because different cells types have various combinations of other gene-regulatory proteins that act with the activated receptor. Together, this variability of intracellular proteins regulates gene transcription in different ways. Cell-surface receptors Cell-surface receptors bind a number of different signalling molecules that cross cell membranes less readily. There are three broad classes of cell-surface receptors that are linked to ion channels, enzymes, or G-proteins. Ligand-gated ion channels are discussed in ‘Cell physiology I’, page 401. On binding a ligand, the receptor (or a nearby membrane protein) acts as an activated ion channel to initiate changes of membrane potential and thereby cellular responses such as initiation or suppression of action potential (see Fry, CROSS REFERENCES). Examples include neurotransmitters such as acetylcholine, glutamate, adenosine triphosphate (ATP) and γ-amino butyric acid. Cell-surface receptors linked to enzymes There are several classes of membrane-bound enzymes with an extracellular ligand-binding site and a cytoplasmic domain with intrinsic enzyme activity or closely associated with an enzyme. The largest classes are the receptor tyrosine kinases
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Receptors linked to guanosine triphosphate (GTP)-binding proteins (G-proteins) The largest group of cell-surface receptors are those that couple to heterotrimeric G-proteins. Most G-protein-linked receptors 408
© 2007 Elsevier Ltd. All rights reserved.
Basic science
• Gi has an α-subunit (αi) that inhibits adenylyl cyclase. (Gi (and a Go group) also activate K+ channels.) • The G-protein associated with phospholipase C-β is Gq. Different G-protein-linked receptors associate with different G-proteins, explaining the diversity of action of extracellular ligands. For example, activation of β-adrenergic receptors activates adenylyl cyclase, while activation of α2-adrenergic receptors inactivates it. αs is the target of cholera toxin (produced by the bacterium Vibrio cholerae) by inhibiting GTPase activity and prolonging the activation of adenylyl cyclase. In the epithelial cells of the gastrointestinal tract, the high concentrations of cAMP cause a large loss of salts and water, resulting in the severe diarrhoea associated with cholera.
initiate a sequence of intracellular events by transiently changing the concentration of small signalling molecules (second messengers), two of which are cyclic adenosine monophosphate (cAMP) and inositol trisphosphate (IP3). G-proteins consist of three subunits: α, β and γ chains. Guanosine diphosphate (GDP) binds to the α-subunit when inactivated. Ligand binding to the G-protein-linked receptor enables the G-protein to associate with it, and to trade GDP for GTP. The α-subunit separates from the β–γ subunit complex and diffuses in the membrane to modulate a target enzyme. • With cAMP, the enzyme is adenylyl cyclase, generating it from ATP. • With IP3, the enzyme is phospholipase C-β, which generates it from a phosphoinositide (PIP2) in the membrane. The lifetime of the activated response is short because the α-subunit hydrolyses GTP, rendering itself inactive. The sequence of events when an extracellular ligand binds to a G-protein-linked receptor is illustrated in Figure 2. Several G-proteins exist, most importantly in relation to the α-subunit. • Gs has an α-subunit (αs) that activates adenylyl cyclase, thereby increasing cAMP concentrations within the cell.
cAMP exerts most of its effects by activating the enzyme protein kinase (A-kinase, PKA). It has diverse actions because the substrates for A-kinase vary in different cells. For example, in skeletal muscle, A-kinase activates phosphorylase kinase and glycogen phosphorylase to increase glycolysis; in myocardium, it increases the conductance of L-type Ca2+ channels, generating a positive inotropic effect. cAMP is metabolized by a group of enzymes called phosphodiesterases, and inhibition of these enzymes prolongs the action of cAMP. For example, phosphodiesterase
Signalling by receptors linked to G-proteins a a Steps in the activation of a receptor linked to a G-protein. In the unbound state (i), the receptor does not influence the trimeric G-protein, to which guanosine diphosphate (GDP) is bound. Ligand binding to the receptor (ii) enables the G-protein to associate with the receptor and enables guanosine triphosphate (GTP) to replace GDP. The α-subunit of the G-protein can dissociate and activate a target enzyme (iii). The process is limited by hydrolysis of GTP and dissociation of the ligand from the receptor. b Targets of the activated α-subunits. The αs subunit activates adenylyl cyclase (AC) to increase the generation of cAMP, which in turn activates protein kinase-A (PKA). The αi/o subunits inhibit adenylyl cyclase, and are also thought to activate K+ channels. The αq subunit activates another enzyme, phospholipase-Cβ (PL-Cβ), which hydrolyses a membrane phospholipid (phosphatidylinositol 4, 5-bisphosphate; PIP2) into two active components: inositol trisphosphate (IP3) and diacylglycerol (DAG). The target for IP2 is receptors on intracellular Ca2+-storing organelles to raise cytosolic [Ca2+]; DAG activates protein kinase-C (PKC). cAMP: Cyclic adenosine phosphate.
Enzyme
Receptor
Cell membrane
βγ
α
αs
GDP
Cell membrane
AC
GTP
cAMP PKA
+
K
Enzyme
Receptor
βγ
Cell membrane
α
αi/o
αi/o
AC
Cell membrane
cAMP
GTP
GDP
b Enzyme
Receptor
βγ
Cell membrane
α
αq
GTP
PL-Cβ
Cell membrane
PIP2 DAG
PKC
IP3
Ca2+
Figure 2
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Basic science
inhibitors have been developed as antihypertensives by generating vasodilation because cAMP causes relaxation of smooth muscle.
junctional adhesion molecules. These molecules interact with cellular scaffold proteins (e.g. ZO-1) which in turn allow attachment to the actin cytoskeleton.
IP3 exerts it effects by diffusing to the endoplasmic (or sarcoplasmic) reticulum, where it binds to receptors that initiate Ca2+ release into the cytoplasm. The lifetime of this response is limited by the breakdown of IP3 by specific phosphatases. Muscarinic M3 receptors are linked to Gq-proteins and their activation explains the action of acetylcholine on initiating contraction in several smooth muscles such as those in the lower urinary tract and gastrointestinal tract. The action of phospholipase C-β on PIP2 generates IP3 and diacylglycerol (DAG). The latter activates protein kinase (PKC), which has several actions on different cells, including regulation of transcription of deoxyribonucleic acid and ion channel function in certain neurones.
Anchoring junctions generate a robust structure to many tissues by mediating cell contacts to neighbouring cells or to the extracellular matrix. They are extensions of the intracellular cyto skeleton and are of several types: • adherens junctions connect actin cytoskeletal filaments to other cells or to the extracellular matrix • desmosomes or hemidesmosomes connect intermediate filaments to neighbouring cells or the extracellular matrix, respectively. Figure 4 shows a model of an adherens junction or a desmosome. The extracellular domains of the junctions (linker proteins) interact with each other, whereas they connect to cytoskeletal elements on the intracellular face. The linker proteins are members of a large family of cadherins (in cell-to-cell contacts), or integ rins (when connected to the extracellular matrix) and require Ca2+ to maintain the contact. In general, adherens junctions lie in a belt of such links, in epithelial cells just below the tight junctions, whereas desmosomes are more punctate and act as rivet connections across the cell surface. Ca2+-independent cell-to-cell adhesion is also possible via cell adhesion molecules, which are derived from the immunoglobulin protein family. The junctions formed by cadherins are stronger than those by cell adhesion molecules, but the latter may regulate specific cell-to-cell contact during development and help in the correct pattern of tissue growth. Interest in these junction proteins is driven by the knowledge that cancerous growths depend upon cell-to-cell contacts and that disruption of this process offers one avenue to attenuate growth.
Cell junctions and the extracellular matrix The collection of cells into tissues requires that cells are positioned precisely in a multicellular array. This is possible by cells being bound together by cellular junctions and the remaining space being occupied by the extracellular matrix, the precise organization differing greatly in various tissues. Types of cell junctions (Table 2) Tight junctions are characteristic of epithelial tissues. Apart from generating structural integrity in the tissue, they separate fluid layers on either side of the epithelial sheet, and permit directional transport of solutes. Figure 3a shows directional transport with reference to reabsorption of Na+ from the proximal tubule of the nephron (see Lote, CROSS REFERENCES). The apical cell surface contains various Na+-linked cotransporters that permit Na+ to enter the cell from the tubular filtrate. The Na+ is removed from the cell by active transporters on the basolateral surface. The positioning of the two sets of transporters allows directional transport of Na+ across the epithelial sheet. The tight junctions prevent the backflow of Na+ by a paracellular route, and also limit the migration of the transporters from their correct places on the apical and basolateral cell surfaces. Tight junctions consist of transmembrane protein arrays that lie near the apical surface of adjacent cells (Figure 3b). The claudins are exclusively responsible for the formation of tight junctions, but other proteins are associated, including occludins and
Gap junctions provide functional coupling between cells by offering cytoplasmic continuity between adjacent cells. A gap junction forms when two connexons from adjacent cells combine; each connexon comprises six connexin proteins that form a central pore. The pore is large because of the six-unit structure of the connexon, and molecules of up to a molecular weight of 1000 can pass through. Chemical signalling is possible between adjacent cells and electric currents can also readily flow between cells because of the cytoplasmic continuity. In tissues like myocardium that have many gap junctions, electrical signals (e.g. action potentials) can propagate between adjacent cells and the entire tissue behaves as a functional syncitium. There are many connexin (Cx) proteins, differentiated by their molecular weight (kiloDaltons), that form pores of different sizes; this is most important because of the electrical continuity they offer: • Cx40 forms large conductance pores, and is abundant in syncitial tissues that offer large action potential conduction velocities (e.g. Purkinje fibres, atrial myocardium) • Cx45 is found in many forms of smooth muscles and offers lower conductance pores. The conductance, or permeability, of gap junctions can be regu lated, and this may underlie important physiological functions. Connexin phosphorylation increases gap junction conductance in some instances and this links metabolic activity and possibly receptor activation to intercellular communication. A rise of cytosolic [Ca2+], or a drop of intracellular pH, reduces conductance;
Cell junctions Type
Action
Tight
Bind cells so tightly even small molecules cannot readily penetrate Form a mechanical attachment between cells, or between a cell and the extracellular matrix Provide a means of intercellular communication
Anchoring Communicating
Table 2
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Basic science
Tight junctions a
b Na+
Na+
Apical Intercellular space Membrane of cell 1 Membrane of cell 2
Tight junctions
Na+
Na+ ZO-1
Basolateral ZO-1 a Unidirectional flux in a cell sheet separated by tight junctions (e.g. proximal tubule of the nephron). There is Na+-coupled flux across the apical membrane by carrier-mediated facilitated diffusion from a region of high to low [Na+]. Na+ is removed from the cell by active transport against a concentration gradient across the basolateral membrane. Na+ is transported across the cell from apical to basolateral side. Backflow between the cells (paracellular movement) is not possible because tight junctions prevent such movement. The tight junctions ensure that the different carriers remain separate (Na+-coupled transporters on apical transport, primary active transporter on the basolateral surface) because they cannot move freely within the plasma membrane between the two faces.
Occludin Claudin Actin
b Tight junction region of adjacent cells. The tight junction proteins form a series of bands that bind the two membranes closely together. The lower box shows the apposition of proteins in the tight junction, including the claudins, occludins and intracellular scaffolding proteins that permit attachment to the cytoskeleton.
Figure 3
this may isolate adjacent cells during cellular dysfunction because this is often associated with these ionic conditions.
Anchoring junctions and gap junctions
The extracellular matrix The space between cells in a tissue comprises the extracellular matrix. The extracellular matrix provides tissue integrity and, in certain tissues: • performs specialized functions (e.g. tendons) • becomes calcified (e.g. bones and teeth) • forms specific structures (e.g. basal lamina below epithelial layers). In most connective tissues—in which the extracellular matrix is particularly plentiful—cells of the fibroblast family (including osteoblasts and chondrocytes) secrete the matrix molecules. These are: • glycosaminoglycans (usually bound to proteins to form proteoglycans) • fibrous proteins (e.g. collagen, elastin) • adhesive proteins (e.g. fibronectin, laminin).
Cytoskeleton
Plaque Cadherin Cell-to-cell desmosome. Two regions of the plasma membranes of adjacent cells. Cadherin molecules from the adjacent cells interact at Ca2+-dependent sites. The cadherin molecules also interact with attachment proteins in intracellular plaques, which in turn link with the cytoskeleton.
Proteoglycans (e.g. chondroitin sulphate, heparan sulphate, keratan sulphate) can be space-filling structures between cells because they can swell by attracting water. They can be
Figure 4
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Basic science
incompressible structures that withstand large compression forces (e.g. cartilage structures). They mediate other functions because of the associated protein moieties: • modulate the function of growth factors (e.g. transforming growth factor-β, fibroblast growth factor) • modulate the function of secreted proteins (e.g. proteases) • act as permeability barriers (e.g. at the nephron glomerulus).
affinity that integrins offer to their ligands ensures that the cell does not bind too strongly to the extracellular matrix and can move within the matrix. This interaction between the cell and the extracellular matrix via integrins has consequences. • Cells that synthesize fewer adhesion molecules are often cancer-like; they can dissociate from a tumour and spread to surrounding tissues. • Blood cells (or fragments of blood cells) require that integrins must be activated only under certain conditions; otherwise they would be less able to circulate freely in the blood. For example, when lymphocytes make weak contact with antigen-producing cells, or platelets contact the wall of a damaged blood vessel, integrins become activated to decrease their mobility and allow them to perform their functions.
Collagens are the most abundant extracellular matrix proteins. They comprise long polypeptide chains that intertwine into a superhelix. Various collagen chains exist, but the most abundant assemble as polymers to form collagen fibrils and fibres and include types I, II and III. Several others, including types IV and VII, form meshes that contribute, for example, to basal lamina structures. Collagen fibrils resist tensile forces and form important components of tendons and skin. Elastin is a protein component of elastic fibres that confer on certain tissues (e.g. blood vessels, lung parenchyma) the ability to stretch and recoil. The integrity of elastic fibres is generated by accessory glycoproteins such as fibrillin. Basal lamina – type IV collagen molecules form an insoluble skeleton to which laminin may be attracted to form a basal lamina. The basal lamina has various functions. It separates epithelial sheets from the bulk of connective tissue and can prevent certain cells (e.g. fibroblasts) but not nerve cells and macrophages from contacting the epithelial cells. In the lung, alveolus and kidney glomerulus, the basal lamina forms an effective molecular permeability barrier to ensure, for example, that plasma proteins do not cross the capillary wall. The importance of the basal lamina also arises during tumour metastasis. In order to spread, tumour cells must disassemble from their neighbours, through weakening cadherin bonds, and cross basal lamina structures by releasing enzymes that facilitate their passage through the extracellular matrix.
Conclusions Cells must communicate with one another so that separate tissue functions are coordinated. Communication within some specialized (excitable) cells is by electrical signalling and such signals may even pass between cells through gap junctions. Chemical signalling between cells is more widespread and may be over a short or long range, and extracellular chemical mediators must find specific receptors to initiate cell responses. Cells must be aligned correctly for tissue integrity, which requires structural intercellular junctions, or interactions between cells and the extracellular matrix. This is achieved by specialized proteins in the cell membrane that link with the cytoskeleton. The breakdown of intercellular communication results in the inability of tissues to communicate adequately, and facilitates many disease processes, including the progression of many cancers. ◆
Cross references Fry C. Action potential and nervous conduction. Surgery 2005; 23(12): 425–9. Lote C. Regulation and disorders of plasma potassium. Surgery 2007; 25(9): 368–74.
The integrins are the principal cell membrane proteins that determine their reaction with the extracellular matrix through binding to collagen, laminin or fibronectin molecules. The relatively low
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Cell physiology II
the resultant effect. Synaptic transmission can be considered a form of paracrine transmission, with the chemical signal termed a ‘neurotransmitter’. Long-distance intercellular signalling utilizes the bloodstream to transport the chemical signal (hormone) from the generating (endocrine) tissue to the target cell. Hormones are examples of ligands that bind to surface and intracellular receptors. Peptide hormones (e.g. antidiuretic hormone) bind to extracellular receptors; steroid hormones (e.g. aldosterone) are lipophilic and readily cross the cell membrane to bind to intracellular receptors.
Christopher Fry
The speed of response varies greatly in the different modes of signalling. • For long-distance signalling, neurotransmission is the most rapid (seconds) because electrical signals convey information once the neurotransmitter has crossed the synaptic cleft. • Hormones that bind to surface receptors have a reasonable response time (minutes) because they initiate cellular responses relatively rapidly once they have reached the target cell. • Hormones that bind to intracellular receptors have the slowest response (hours) because they modulate transcription of deoxyribonucleic acid.
Abstract For multicellular organisms, specialization of function has evolved to enable different tissues to carry out specific tasks crucial to the survival of the entire organism. Communication between different cells is vital to coordinate the activity of these separate functions. Individual cells within a tissue must be oriented appropriately to fulfil organ function and structural integrity of the tissue must be maintained. This contribution considers how such group activity is achieved.
Keywords cell signalling; calcium; receptors; autocrine; intracellular; cell- surface; enzymes; G-proteins; cell junctions; tight junctions; anchoring junctions; gap junctions; extracellular; proteoglycans; collagens; integrins
Autocrine signalling: allied to intercellular signalling is the ability of some cells to influence their own activity through the release of chemical signalling molecules. Such autocrine signalling may be a positive feedback process, whereby the release of a particular ligand may accelerate a particular cell function, including a greater release of the ligand.
Cell signalling Basic concepts of cell signalling Type of signal: cells can communicate via chemical mediators or by electrical signals (the latter are considered below with respect to gap junctions). Chemical – in general, the target cell responds to chemical signals through a receptor that often has a very high affinity for a particular signalling molecule. A few signalling molecules can cross the cell membrane easily and directly regulate intracellular reactions. An example is the volatile gas nitric oxide, produced by the action of nitric oxide synthase, the activity of which is modulated by Ca2+ released in response to agents such as acetylcholine. Nitric oxide activates guanylyl cyclase to produce cyclic guanosine monophosphate to generate cellular responses such as relaxation of smooth muscles, as in vascular tissue or the corpus cavernosum.
Differential response of ligands: a particular ligand may generate different responses in different cells. This is because receptors to a particular ligand can be of several types and subtypes that elicit different intracellular responses; also, a particular subtype may initiate different responses in various cells. An example is the ligand acetylcholine, which can bind to nicotinic or muscarinic receptors and can mediate a variety of responses (Table 1). Intracellular receptors Intracellular receptors are a group of transcription factors that depend upon interaction with ligands: they include steroid hormones (including the mineralocorticoids, glucocorticoids and sex hormones), vitamin D and thyroid hormone. Figure 1a shows the general scheme: a lipophilic signalling agent diffuses into the cell, binds to receptors and initiates a series of steps to generate products that mediate the response of the hormone. Different cell types may have the same steroid receptor, but generate dissimilar
Receptors are proteins on the cell surface that initiate cellular reactions when a chemical (ligand) binds; or they are intracellular receptors that require the ligand to cross the cell membrane. Some signalling molecules act locally and must be rapidly removed or immobilized so that they do not to travel too far. An example of such a paracrine mediator is histamine, released from mast cells and basophils when sensitized by antibodies to generate local effects such as vasodilation or bronchoconstriction. Such local action is not sufficient in larger multicellular organisms, and target cells such as nerves have developed long axons to propagate
Effects of acetylcholine • Nervous transmission at autonomic ganglia • Excitation of skeletal muscle at the motor endplate • Contraction of various smooth muscles • Slowing of pacemaker rate at the sinoatrial node • Stimulation of secretion by salivary glands
Christopher Fry DSc is the Professor of Cell Physiology at Royal Free Hospital, London, UK. Conflicts of interest: none declared.
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Table 1
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© 2007 Elsevier Ltd. All rights reserved.
Basic science
Signalling by intracellular receptors and surface enzyme receptors
a
Steroid
b GTP Activated Ras
MAPKKK (Raf)
Ligand Primary response
Intracellular receptor
GNRP
MAPKK (MEK 1/2)
Receptor tyrosine kinase
DNA
MAPK (ERK 1/2) GAP GDP
Secondary response products
a Signalling by lipophilic agents, typified by a steroid hormone. The steroid diffuses into the cell and binds to an intracellular receptor. This complex facilitates transcription to generate several primary products. These in turn initiate transcription of genes for a number of secondary products that mediate the cellular response, and limit the production of further primary products. The same ligand and intracellular receptor regulate the transcription of different genes in different cells, enabling specificity of action. DNA: Deoxyribonucleic acid.
Inactive Ras
Target proteins in nucleus
Target proteins in cytoplasm
Cell membrane b Steps in the mitogen-activated pathway. A receptor tyrosine kinase (RTK) binds a ligand (e.g. growth factor) and initiates a series of intracellular steps that starts with activation of a membrane-bound monomeric GTPase, Ras. Ras is active when it binds guanosine triphosphate (GTP) and inactive when it binds guanosine diphosphate (GDP); the switch between the two states can be governed by GTPase-activating proteins (GAPs) and guanine nucleotide release proteins (GNRPs). RTK can activate Ras by activating a GNRP or by inactivating a GAP. Once activated, Ras initiates a further series of phosphorylations, culminating in activation of a mitogen-activated protein kinase (MAPK), which can activate nuclear and cytoplasmic proteins to initiate cellular events. Some MAPKs are known, including the extracellular signal-regulated kinases (ERKs). The intermediate steps in the MAP kinase pathways involve successive kinases; MAPK kinases (e.g. MEKs – MAP-ERK kinases); and MAPK kinase-kinases (e.g. Raf).
Figure 1
and tyrosine- associated receptor kinases. Ligands include many growth and differentiation factors, such as: • epidermal and nerve growth factors • vascular endothelial growth factor • platelet-derived growth factor. Upon activation, the receptors transfer a phosphate group from ATP to specific tyrosine moieties on the intracellular domain of the receptor or closely associated proteins. Many cytoplasmic proteins bind to the phosphorylated tyrosines to initiate cellular responses, including the Ras proteins, which stimulate proliferation and differentiation of cells. Figure 1b shows that a cascade of reactions involves mitogen-activated protein kinases and activation of regulatory gene proteins. Other enzyme-linked receptor pathways include the serine/ threonine protein kinases, which bind transforming growth factor-β proteins that initiate similar intracellular pathways through the mediation of Smad proteins. The receptor guanylyl cyclases, which bind atrial natriuretic peptides, generate cyclic guanosine monophosphate to mediate intracellular responses.
cellular responses. This is because different cells types have various combinations of other gene-regulatory proteins that act with the activated receptor. Together, this variability of intracellular proteins regulates gene transcription in different ways. Cell-surface receptors Cell-surface receptors bind a number of different signalling molecules that cross cell membranes less readily. There are three broad classes of cell-surface receptors that are linked to ion channels, enzymes, or G-proteins. Ligand-gated ion channels are discussed in ‘Cell physiology I’, page 401. On binding a ligand, the receptor (or a nearby membrane protein) acts as an activated ion channel to initiate changes of membrane potential and thereby cellular responses such as initiation or suppression of action potential (see Fry, CROSS REFERENCES). Examples include neurotransmitters such as acetylcholine, glutamate, adenosine triphosphate (ATP) and γ-amino butyric acid. Cell-surface receptors linked to enzymes There are several classes of membrane-bound enzymes with an extracellular ligand-binding site and a cytoplasmic domain with intrinsic enzyme activity or closely associated with an enzyme. The largest classes are the receptor tyrosine kinases
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Receptors linked to guanosine triphosphate (GTP)-binding proteins (G-proteins) The largest group of cell-surface receptors are those that couple to heterotrimeric G-proteins. Most G-protein-linked receptors 408
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Basic science
• Gi has an α-subunit (αi) that inhibits adenylyl cyclase. (Gi (and a Go group) also activate K+ channels.) • The G-protein associated with phospholipase C-β is Gq. Different G-protein-linked receptors associate with different G-proteins, explaining the diversity of action of extracellular ligands. For example, activation of β-adrenergic receptors activates adenylyl cyclase, while activation of α2-adrenergic receptors inactivates it. αs is the target of cholera toxin (produced by the bacterium Vibrio cholerae) by inhibiting GTPase activity and prolonging the activation of adenylyl cyclase. In the epithelial cells of the gastrointestinal tract, the high concentrations of cAMP cause a large loss of salts and water, resulting in the severe diarrhoea associated with cholera.
initiate a sequence of intracellular events by transiently changing the concentration of small signalling molecules (second messengers), two of which are cyclic adenosine monophosphate (cAMP) and inositol trisphosphate (IP3). G-proteins consist of three subunits: α, β and γ chains. Guanosine diphosphate (GDP) binds to the α-subunit when inactivated. Ligand binding to the G-protein-linked receptor enables the G-protein to associate with it, and to trade GDP for GTP. The α-subunit separates from the β–γ subunit complex and diffuses in the membrane to modulate a target enzyme. • With cAMP, the enzyme is adenylyl cyclase, generating it from ATP. • With IP3, the enzyme is phospholipase C-β, which generates it from a phosphoinositide (PIP2) in the membrane. The lifetime of the activated response is short because the α-subunit hydrolyses GTP, rendering itself inactive. The sequence of events when an extracellular ligand binds to a G-protein-linked receptor is illustrated in Figure 2. Several G-proteins exist, most importantly in relation to the α-subunit. • Gs has an α-subunit (αs) that activates adenylyl cyclase, thereby increasing cAMP concentrations within the cell.
cAMP exerts most of its effects by activating the enzyme protein kinase (A-kinase, PKA). It has diverse actions because the substrates for A-kinase vary in different cells. For example, in skeletal muscle, A-kinase activates phosphorylase kinase and glycogen phosphorylase to increase glycolysis; in myocardium, it increases the conductance of L-type Ca2+ channels, generating a positive inotropic effect. cAMP is metabolized by a group of enzymes called phosphodiesterases, and inhibition of these enzymes prolongs the action of cAMP. For example, phosphodiesterase
Signalling by receptors linked to G-proteins a a Steps in the activation of a receptor linked to a G-protein. In the unbound state (i), the receptor does not influence the trimeric G-protein, to which guanosine diphosphate (GDP) is bound. Ligand binding to the receptor (ii) enables the G-protein to associate with the receptor and enables guanosine triphosphate (GTP) to replace GDP. The α-subunit of the G-protein can dissociate and activate a target enzyme (iii). The process is limited by hydrolysis of GTP and dissociation of the ligand from the receptor. b Targets of the activated α-subunits. The αs subunit activates adenylyl cyclase (AC) to increase the generation of cAMP, which in turn activates protein kinase-A (PKA). The αi/o subunits inhibit adenylyl cyclase, and are also thought to activate K+ channels. The αq subunit activates another enzyme, phospholipase-Cβ (PL-Cβ), which hydrolyses a membrane phospholipid (phosphatidylinositol 4, 5-bisphosphate; PIP2) into two active components: inositol trisphosphate (IP3) and diacylglycerol (DAG). The target for IP2 is receptors on intracellular Ca2+-storing organelles to raise cytosolic [Ca2+]; DAG activates protein kinase-C (PKC). cAMP: Cyclic adenosine phosphate.
Enzyme
Receptor
Cell membrane
βγ
α
αs
GDP
Cell membrane
AC
GTP
cAMP PKA
+
K
Enzyme
Receptor
βγ
Cell membrane
α
αi/o
αi/o
AC
Cell membrane
cAMP
GTP
GDP
b Enzyme
Receptor
βγ
Cell membrane
α
αq
GTP
PL-Cβ
Cell membrane
PIP2 DAG
PKC
IP3
Ca2+
Figure 2
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inhibitors have been developed as antihypertensives by generating vasodilation because cAMP causes relaxation of smooth muscle.
junctional adhesion molecules. These molecules interact with cellular scaffold proteins (e.g. ZO-1) which in turn allow attachment to the actin cytoskeleton.
IP3 exerts it effects by diffusing to the endoplasmic (or sarcoplasmic) reticulum, where it binds to receptors that initiate Ca2+ release into the cytoplasm. The lifetime of this response is limited by the breakdown of IP3 by specific phosphatases. Muscarinic M3 receptors are linked to Gq-proteins and their activation explains the action of acetylcholine on initiating contraction in several smooth muscles such as those in the lower urinary tract and gastrointestinal tract. The action of phospholipase C-β on PIP2 generates IP3 and diacylglycerol (DAG). The latter activates protein kinase (PKC), which has several actions on different cells, including regulation of transcription of deoxyribonucleic acid and ion channel function in certain neurones.
Anchoring junctions generate a robust structure to many tissues by mediating cell contacts to neighbouring cells or to the extracellular matrix. They are extensions of the intracellular cyto skeleton and are of several types: • adherens junctions connect actin cytoskeletal filaments to other cells or to the extracellular matrix • desmosomes or hemidesmosomes connect intermediate filaments to neighbouring cells or the extracellular matrix, respectively. Figure 4 shows a model of an adherens junction or a desmosome. The extracellular domains of the junctions (linker proteins) interact with each other, whereas they connect to cytoskeletal elements on the intracellular face. The linker proteins are members of a large family of cadherins (in cell-to-cell contacts), or integ rins (when connected to the extracellular matrix) and require Ca2+ to maintain the contact. In general, adherens junctions lie in a belt of such links, in epithelial cells just below the tight junctions, whereas desmosomes are more punctate and act as rivet connections across the cell surface. Ca2+-independent cell-to-cell adhesion is also possible via cell adhesion molecules, which are derived from the immunoglobulin protein family. The junctions formed by cadherins are stronger than those by cell adhesion molecules, but the latter may regulate specific cell-to-cell contact during development and help in the correct pattern of tissue growth. Interest in these junction proteins is driven by the knowledge that cancerous growths depend upon cell-to-cell contacts and that disruption of this process offers one avenue to attenuate growth.
Cell junctions and the extracellular matrix The collection of cells into tissues requires that cells are positioned precisely in a multicellular array. This is possible by cells being bound together by cellular junctions and the remaining space being occupied by the extracellular matrix, the precise organization differing greatly in various tissues. Types of cell junctions (Table 2) Tight junctions are characteristic of epithelial tissues. Apart from generating structural integrity in the tissue, they separate fluid layers on either side of the epithelial sheet, and permit directional transport of solutes. Figure 3a shows directional transport with reference to reabsorption of Na+ from the proximal tubule of the nephron (see Lote, CROSS REFERENCES). The apical cell surface contains various Na+-linked cotransporters that permit Na+ to enter the cell from the tubular filtrate. The Na+ is removed from the cell by active transporters on the basolateral surface. The positioning of the two sets of transporters allows directional transport of Na+ across the epithelial sheet. The tight junctions prevent the backflow of Na+ by a paracellular route, and also limit the migration of the transporters from their correct places on the apical and basolateral cell surfaces. Tight junctions consist of transmembrane protein arrays that lie near the apical surface of adjacent cells (Figure 3b). The claudins are exclusively responsible for the formation of tight junctions, but other proteins are associated, including occludins and
Gap junctions provide functional coupling between cells by offering cytoplasmic continuity between adjacent cells. A gap junction forms when two connexons from adjacent cells combine; each connexon comprises six connexin proteins that form a central pore. The pore is large because of the six-unit structure of the connexon, and molecules of up to a molecular weight of 1000 can pass through. Chemical signalling is possible between adjacent cells and electric currents can also readily flow between cells because of the cytoplasmic continuity. In tissues like myocardium that have many gap junctions, electrical signals (e.g. action potentials) can propagate between adjacent cells and the entire tissue behaves as a functional syncitium. There are many connexin (Cx) proteins, differentiated by their molecular weight (kiloDaltons), that form pores of different sizes; this is most important because of the electrical continuity they offer: • Cx40 forms large conductance pores, and is abundant in syncitial tissues that offer large action potential conduction velocities (e.g. Purkinje fibres, atrial myocardium) • Cx45 is found in many forms of smooth muscles and offers lower conductance pores. The conductance, or permeability, of gap junctions can be regu lated, and this may underlie important physiological functions. Connexin phosphorylation increases gap junction conductance in some instances and this links metabolic activity and possibly receptor activation to intercellular communication. A rise of cytosolic [Ca2+], or a drop of intracellular pH, reduces conductance;
Cell junctions Type
Action
Tight
Bind cells so tightly even small molecules cannot readily penetrate Form a mechanical attachment between cells, or between a cell and the extracellular matrix Provide a means of intercellular communication
Anchoring Communicating
Table 2
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Tight junctions a
b Na+
Na+
Apical Intercellular space Membrane of cell 1 Membrane of cell 2
Tight junctions
Na+
Na+ ZO-1
Basolateral ZO-1 a Unidirectional flux in a cell sheet separated by tight junctions (e.g. proximal tubule of the nephron). There is Na+-coupled flux across the apical membrane by carrier-mediated facilitated diffusion from a region of high to low [Na+]. Na+ is removed from the cell by active transport against a concentration gradient across the basolateral membrane. Na+ is transported across the cell from apical to basolateral side. Backflow between the cells (paracellular movement) is not possible because tight junctions prevent such movement. The tight junctions ensure that the different carriers remain separate (Na+-coupled transporters on apical transport, primary active transporter on the basolateral surface) because they cannot move freely within the plasma membrane between the two faces.
Occludin Claudin Actin
b Tight junction region of adjacent cells. The tight junction proteins form a series of bands that bind the two membranes closely together. The lower box shows the apposition of proteins in the tight junction, including the claudins, occludins and intracellular scaffolding proteins that permit attachment to the cytoskeleton.
Figure 3
this may isolate adjacent cells during cellular dysfunction because this is often associated with these ionic conditions.
Anchoring junctions and gap junctions
The extracellular matrix The space between cells in a tissue comprises the extracellular matrix. The extracellular matrix provides tissue integrity and, in certain tissues: • performs specialized functions (e.g. tendons) • becomes calcified (e.g. bones and teeth) • forms specific structures (e.g. basal lamina below epithelial layers). In most connective tissues—in which the extracellular matrix is particularly plentiful—cells of the fibroblast family (including osteoblasts and chondrocytes) secrete the matrix molecules. These are: • glycosaminoglycans (usually bound to proteins to form proteoglycans) • fibrous proteins (e.g. collagen, elastin) • adhesive proteins (e.g. fibronectin, laminin).
Cytoskeleton
Plaque Cadherin Cell-to-cell desmosome. Two regions of the plasma membranes of adjacent cells. Cadherin molecules from the adjacent cells interact at Ca2+-dependent sites. The cadherin molecules also interact with attachment proteins in intracellular plaques, which in turn link with the cytoskeleton.
Proteoglycans (e.g. chondroitin sulphate, heparan sulphate, keratan sulphate) can be space-filling structures between cells because they can swell by attracting water. They can be
Figure 4
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incompressible structures that withstand large compression forces (e.g. cartilage structures). They mediate other functions because of the associated protein moieties: • modulate the function of growth factors (e.g. transforming growth factor-β, fibroblast growth factor) • modulate the function of secreted proteins (e.g. proteases) • act as permeability barriers (e.g. at the nephron glomerulus).
affinity that integrins offer to their ligands ensures that the cell does not bind too strongly to the extracellular matrix and can move within the matrix. This interaction between the cell and the extracellular matrix via integrins has consequences. • Cells that synthesize fewer adhesion molecules are often cancer-like; they can dissociate from a tumour and spread to surrounding tissues. • Blood cells (or fragments of blood cells) require that integrins must be activated only under certain conditions; otherwise they would be less able to circulate freely in the blood. For example, when lymphocytes make weak contact with antigen-producing cells, or platelets contact the wall of a damaged blood vessel, integrins become activated to decrease their mobility and allow them to perform their functions.
Collagens are the most abundant extracellular matrix proteins. They comprise long polypeptide chains that intertwine into a superhelix. Various collagen chains exist, but the most abundant assemble as polymers to form collagen fibrils and fibres and include types I, II and III. Several others, including types IV and VII, form meshes that contribute, for example, to basal lamina structures. Collagen fibrils resist tensile forces and form important components of tendons and skin. Elastin is a protein component of elastic fibres that confer on certain tissues (e.g. blood vessels, lung parenchyma) the ability to stretch and recoil. The integrity of elastic fibres is generated by accessory glycoproteins such as fibrillin. Basal lamina – type IV collagen molecules form an insoluble skeleton to which laminin may be attracted to form a basal lamina. The basal lamina has various functions. It separates epithelial sheets from the bulk of connective tissue and can prevent certain cells (e.g. fibroblasts) but not nerve cells and macrophages from contacting the epithelial cells. In the lung, alveolus and kidney glomerulus, the basal lamina forms an effective molecular permeability barrier to ensure, for example, that plasma proteins do not cross the capillary wall. The importance of the basal lamina also arises during tumour metastasis. In order to spread, tumour cells must disassemble from their neighbours, through weakening cadherin bonds, and cross basal lamina structures by releasing enzymes that facilitate their passage through the extracellular matrix.
Conclusions Cells must communicate with one another so that separate tissue functions are coordinated. Communication within some specialized (excitable) cells is by electrical signalling and such signals may even pass between cells through gap junctions. Chemical signalling between cells is more widespread and may be over a short or long range, and extracellular chemical mediators must find specific receptors to initiate cell responses. Cells must be aligned correctly for tissue integrity, which requires structural intercellular junctions, or interactions between cells and the extracellular matrix. This is achieved by specialized proteins in the cell membrane that link with the cytoskeleton. The breakdown of intercellular communication results in the inability of tissues to communicate adequately, and facilitates many disease processes, including the progression of many cancers. ◆
Cross references Fry C. Action potential and nervous conduction. Surgery 2005; 23(12): 425–9. Lote C. Regulation and disorders of plasma potassium. Surgery 2007; 25(9): 368–74.
The integrins are the principal cell membrane proteins that determine their reaction with the extracellular matrix through binding to collagen, laminin or fibronectin molecules. The relatively low
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