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Membranes and 5 Intracellular Signal Transduction The phospholipids contain two fatty acids (usually 16 to 18 CONTENTS...

Membranes and 5 Intracellular Signal Transduction The phospholipids contain two fatty acids (usually 16 to 18 CONTENTS carbons) attached to glycerol in addition to a phosphate MEMBRANE STRUCTURE AND COMPOSITION group. The fatty acids may be either unsaturated or saturated. Membrane Components Most phospholipids have ethanolamine, choline, inositol, or Membrane Structure serine esterified to the phosphate. Fluid Properties of Membranes The sphingolipids include sphingomyelin, cerebrosides, MEMBRANE TRANSPORT and gangliosides. The cerebrosides and gangliosides, sugar- Simple Diffusion containing lipids called glycosphingolipids, are located pri- Facilitated Diffusion Active Transport marily in the plasma membrane. Sphingomyelin is prominent INTRACELLULAR SIGNAL TRANSDUCTION in myelin sheaths. Plasma Membrane Receptors Cholesterol is primarily found in the plasma membrane Cyclic Adenosine Monophosphate System—Epinephrine with its hydroxyl group on the surface at the water interface. and Glucagon Membranes are generally 40% to 50% protein but can G-Protein–Mediated Signal Transduction range from extremes such as 20% protein in the myelin Desensitization to Epinephrine membrane to 80% protein in the inner mitochondrial mem- Phosphoinositide Cascade brane. Protein and lipid composition is unique for each Tyrosine Kinase Receptors membrane, and their distribution is asymmetric. Nitric Oxide and Cyclic Guanosine Monophosphate Intracellular Receptors of Lipophilic Hormones Clinical Aspects of Intracellular Signaling Integral Membrane Proteins Integral membrane proteins may penetrate the membrane partially or may exist as transmembrane proteins interfacing with both the cytosol and external environment. lll MEMBRANE STRUCTURE They interact strongly with the membrane lipids through AND COMPOSITION hydrophobic side chains of amino acids and can only be re- moved by destroying membrane structure with detergent or Membranes are composed of various lipids, proteins, and car- solvent. They are usually composed of multiple a-helices with bohydrates that determine several important biologic func- hydrophobic side chains; cylindrical arrays form pores for tions. Their selective permeability affords both a physical transport of polar molecules. and chemical compartmentation of intracellular enzyme sys- tems. Membranes also contain enzymes and receptors that allow cells to respond selectively to external signals, as well Peripheral Membrane Proteins Peripheral membrane proteins are loosely associated with the as to generate chemical and electrical signals. Their selective surface of either side of the membrane; they interact with permeability is regulated by molecular channels and pumps the membrane through hydrogen bonding or salt-bridging that extend between the two surfaces. Their external surface with membrane proteins or lipids and can be removed without composition determines cell-to-cell recognition processes disrupting the structure of the membrane. mediating cell adhesion and immune responses. Membrane carbohydrates exist only as extracellular cova- lent attachments to lipids and proteins (e.g., glycoproteins Membrane Components or glycolipids). Carbohydrate structures are highly variable The membrane lipids include phospholipids, sphingolipids, and may be highly antigenic, thereby contributing to the im- and cholesterol (see Chapter 11). mune recognition of cells.  40 Membranes and Intracellular Signal Transduction Membrane Structure diffusion in membrane. Transverse diffusion is energetically very unfavorable; neither proteins nor lipids “flip-flop” from Membranes achieve their selective permeability by separation one side to the other, except when the process is catalyzed by of the internal and external aqueous compartments with a enzymes called flippases. phospholipid bilayer. The bilayer is formed from two mono- Fluidity is affected by several factors: layers, or leaflets, composed of phospholipids with the hydro- l Long-chain saturated fatty acids interact strongly and philic phosphate head groups oriented toward the aqueous reduce fluidity. solution and the hydrophobic fatty acid tails oriented toward l Double bonds increase fluidity, greater with cis-configuration the center of the bilayer (Fig. 5-1). The bilayers form sheetlike than with trans-configuration. structures measuring between 60 and 100 Å in thickness and l Cholesterol prevents movement of fatty acid chains and are held together entirely by noncovalent forces. reduces fluidity. Although the bilayer structure is symmetric with respect to l Fluidity increases with temperature. orientation of the amphipathic lipids (containing both hydro- philic and hydrophobic regions), the composition is asymmet- ric. For example, the red blood cell plasma membrane has the KEY POINTS ABOUT MEMBRANE STRUCTURE following phospholipid composition: AND COMPOSITION l Exterior monolayer: mostly sphingomyelin and phosphati- n Membranes serve several important functions: compartmenta- dylcholine tion of enzyme systems, receptor recognition of hormone signals, l Interior monolayer: mostly phosphatidylserine and phos- generation of chemical and electrical signals, selective transport phatidylethanolamine of molecules, and cell-to-cell recognition and adhesion. Cholesterol is more evenly distributed, with the precise n Membranes contain lipid, protein, and carbohydrate arranged in composition determined by the function of the membrane. a fluid mosaic bilayer leaflet. Membrane proteins are distributed asymmetrically to pro- vide localization of enzyme activity, energy transduction n Membrane lipids include phospholipids, sphingolipids, and through ion pumps, facilitated transport, and receptors for cholesterol. extracellular signals. Peripheral proteins often contain a lipid n Membrane proteins make up between 20% and 80% of a given anchor that extends into the membrane. membrane, but typically 40% to 50%. Membrane composition and asymmetry are maintained by n Integral membrane proteins are hydrophobic and cannot be iso- addition of new membrane structure to preexisting membrane lated without destroying the membrane; peripheral membrane structure. Self-assembly permits self-sealing of damage to the proteins are associated only with the surface and can be removed phospholipid bilayer. easily. n Membrane carbohydrate is found on the external surface attached to proteins and lipids and helps determine immune Fluid Properties of Membranes recognition of cells. The assembly of proteins and lipids into a membrane creates a n Membrane proteins and lipids are distributed asymmetrically and fluid mosaic, named for the fluid properties of its constituents. undergo lateral diffusion only. Both the proteins and lipids undergo two-dimensional lateral Carbohydrate chain Cholesterol Phospholipid Peripheral proteins Integral protein Figure 5-1. Membrane components.  Membrane transport 41 lll MEMBRANE TRANSPORT l Dissociation constant for the transported molecule, Tm, analogous to Km for enzymes. The hydrophobic property of membranes effectively blocks the l Inhibition by agents that block the transport of specific movement of hydrophilic molecules across the membrane. Fur- molecules. thermore, the structural integrity of the membrane restricts dif- l Exhibited saturation kinetics (Vmax). fusion through the lipid bilayer. Thus movement of molecules Facilitated diffusion has three primary modes: ion channels, across the membrane is governed by several forms of transport: uniporters, and cotransporters. simple diffusion, facilitated diffusion, and active transport. Ion channels are protein-lined channels that selectively al- low ions to flow at a high rate when they are open. The ion Simple Diffusion channel is formed by multiple transmembrane domains of the specific ion channel protein. Some channels are referred Water, gases (O2, CO2, NO), and lipophilic molecules (small to as “gated,” since they are only opened transiently in re- fatty acids, steroids, urea, ethanol) cross membranes by sim- sponse to specific signals. The signal for a ligand-gated channel ple diffusion. Simple diffusion always occurs down a concen- is the binding of the specific ligand to a receptor. Voltage- tration gradient and may be in either direction, depending gated channels respond to changes in the membrane potential. only on the direction of the gradient. A steep concentration Uniporters (Fig. 5-3) facilitate diffusion of single substances, gradient produces faster diffusion than a shallow gradient, such as glucose or a specific amino acid. The GLUT family of and smaller molecules diffuse faster than larger molecules. sodium-independent glucose transporters are uniporters that Simple diffusion is not saturable (i.e., the rate of diffusion in- passively transport glucose (and/or galactose and fructose) creases linearly with the increase in substrate concentration into most cells. Alternative conformations of the transporter gradient across the membrane). allow binding at the exterior surface (high glucose concentra- tion) and release at the interior surface (low glucose concen- tration). Also, the discovery of aquaporins shows that water Facilitated Diffusion can also enter by facilitated diffusion. When molecules are excluded from simple diffusion owing to Cotransporters transport more than one molecule simulta- size or charge, facilitated diffusion mechanisms exist. Special- neously (see Fig. 5-3). Symporters carry two different mole- ized carrier proteins in the membrane either diffuse across the cules in the same direction at the same time, whereas membrane with their substrate or extend across the mem- antiporters carry two different molecules in opposite directions brane, forming a channel. at the same time. Similarities to simple diffusion: An example of a symporter is found in the kidney and intes- l Diffusion occurs down a concentration gradient. tine, where glucose must be transported from the lumen l Energy is supplied by the gradient, not by cellular energy. into the cell against a concentration gradient. The sodium- Differences from simple diffusion: dependent glucose symporter relies on a gradient generated l Facilitated diffusion is faster than simple diffusion. by active transport of sodium out of the cell (Fig. 5-4), then l The carrier has specificity for the transported substance. the downhill transport of sodium is coupled to the uphill trans- l Facilitated diffusion displays saturation (hyperbolic) kinetics port of glucose. (Fig. 5-2). An example of an antiporter is the chloride-bicarbonate Carrier proteins are variously called translocases, porters, transporter in erythrocyte membranes. Bicarbonate must and permeases; their similarity to enzymes is shown by the undergo compensating transport with CO2 (i.e., CO2 in, following: HCO3– out). This is mediated by the chloride-bicarbonate ex- l Structural specificity for transported molecules. changer (Fig. 5-5). Bicarbonate transport is accompanied by S S1 S2 S1 Vmax Rate 1/ 2 Vmax Carrier-mediated transport Passive diffusion Concentration S2 Km Uniport Symport Antiport Figure 5-2. Comparison of carrier-mediated transport with pas- Cotransport sive diffusion. Carrier-mediated transport can reach saturation kinetics. Figure 5-3. Uniport vs. cotransport.  42 Membranes and Intracellular Signal Transduction ATP ADP+Pi KEY POINTS ABOUT MEMBRANE TRANSPORT n Lipophilic molecules, including small, uncharged molecules such Glucose Na; 3 Na; 2 K; as water and oxygen, diffuse through membranes by simple diffu- sion down a concentration gradient. Cytoplasm n Specialized carrier proteins facilitate the diffusion of many mole- cules; they are specific for the molecule transported and move down a concentration gradient. n Facilitated diffusion may involve more than one molecule in one direction (uniport), so that two molecules are exchanged (anti- port) or transported together (symport). Lumen n Active transport against a gradient is accomplished by coupling transport with ATP hydrolysis. Glucose Na; 3 Na; 2 K; Figure 5-4. Sodium-dependent glucose transporter. The Naþ/Kþ adenosine triphosphate (ATP) pump maintains an Naþ gradient for lll INTRACELLULAR SIGNAL cotransport with glucose. ADP, adenosine diphosphate. TRANSDUCTION Hormones are physiologic signals that influence cellular me- Cl– transport in the opposite direction (antiport) to maintain tabolism by triggering a sequence of coordinated intracellular electrical neutrality. Like other antiporters, the chloride- responses. The conversion of the signal from the hormone bicarbonate transporter works in either direction as determined molecule to the final change in activity of the target enzymes by the concentration gradient (lungs or tissues). is transmitted (transduced) through a signal transduction cascade. Since each reaction step in the cascade produces a Active Transport catalyst as its product, each step in the cascade serves to am- Carrier proteins that transport molecules against a gradient plify the signal. The signal can be lipophilic or hydrophilic can be directly coupled to hydrolysis of adenosine triphos- (Table 5-1). phate (ATP) (i.e., hydrolysis of ATP provides the energy to drive the uphill transport process). This process is called active transport and is unidirectional. Like facilitated diffusion it is Plasma Membrane Receptors specific for the molecules being transported, it demonstrates Plasma membrane receptors are transmembrane proteins that saturation kinetics, and it can be specifically inhibited. Since generate an intracellular response following binding of hor- it is tightly coupled to the hydrolysis of ATP, there is no mones, cytokines, and other signals on the exterior surface ATP hydrolysis without transport. of the cell. They share several characteristics with enzymes: The sodium/potassium ATPase (Naþ/Kþ-ATPase) antipor- l Hormone binding induces a conformational change in the ter is an example of active transport. This active transport receptor protein (such as allosteric regulation). pump is located in the plasma membrane of every cell. It l Hormone binding demonstrates reversibility (such as the maintains low intracellular Naþ and high intracellular Kþ. enzyme-substrate complex). This antiporter pumps 3 Naþ out and 2 Kþ in for every l Hormone binding demonstrates inhibition (by antagonists; ATP hydrolyzed (see Fig. 5-5). competitive or noncompetitive kinetics). Erythrocyte in the Tissues Erythrocyte in the Lungs HCO3 : Cl: CO2 Carbonic Carbonic anhydrase anhydrase CO2 + H2O HCO3: + H; Cl: CO2 + H2O HCO3: + H; Cl: CO2 HCO3: Cl: Figure 5-5. Chloride-bicarbonate transporter. Chloride is exchanged for bicarbonate to “pull” CO2 from the tissues into the red blood cell. Reversal in the lungs allows for expiration of CO2.  Intracellular signal transduction 43 TABLE 5-1. Hormone Signals CHARACTERISTIC HYDROPHILIC LIPOPHILIC Intracellular messenger cAMP, cGMP, phosphoinositides, Hormone-receptor complex diacylglycerol, Caþþ Duration of action Minutes Hours to days Location of receptor Plasma membrane Intracellular Type of hormone Polypeptide hormones, growth factors, cytokines Steroids, retinoids, calcitriol, thyroxine The response to a given hormone can be positive or nega- tive depending on which receptors are present. Receptor- Glucagon hormone dissociation constants correlate with the physiologic concentrations of the hormones. Only a small fraction of the receptors needs to be occupied to provide an effective response. Exterior PHARMACOLOGY Cytosol Cardiotonic Steroids Digoxin and digitoxin (cardiotonic steroids) affect heart ATP contractility by inhibiting Naþ/Kþ-ATPase. This inhibits the cAMP AMP calcium-sodium transporter, causing intracellular calcium to + increase. The increased intracellular calcium augments myocardial contractility, producing a cardiotonic effect. ATP Protein kinase A ADP Protein Phosphoprotein Cyclic Adenosine Monophosphate System—Epinephrine and Glucagon Protein phosphatase When epinephrine and glucagon bind to their receptors, they send a wave of phosphorylation through the cell that leads to Figure 5-7. Activation of protein kinase A by cyclic adenosine coordinated changes in metabolism. The initial signal that is monophosphate (cAMP). ATP, adenosine triphosphate; ADP, generated in this pathway is the second messenger molecule, adenosine diphosphate. cyclic adenosine monophosphate (cAMP). cAMP is synthe- sized by a membrane-bound adenylate cyclase when the PATHOLOGY hormone binds to the receptor (Fig. 5-6). The concentration of cAMP is determined by the balance between adenylate Cystic Fibrosis cyclase and cAMP phosphodiesterase activity that degrades Cystic fibrosis is due to a defective chloride ATPase pump in the cAMP to AMP. the epithelial cells of the lungs, intestines, skin, and pancreas. The increased concentration of cAMP, in turn, allosterically This leads to very high concentrations of Naþ and Cl– in sweat converts more protein kinase A to its active form (Fig. 5-7). and the production of highly viscous mucus that obstructs the Protein kinase A regulates a variety of target proteins and pancreatic and bile ducts and the airways in the lungs. enzymes by phosphorylation with ATP. 2 Pi 5„ P J P J P JCH2 O Adenine P~Pi CH2 O Adenine P JCH2 O Adenine J O Adenylate cAMP cyclase 3„ phosphodiesterase OH OH :O JPJO OH OH OH K ATP O cAMP AMP Figure 5-6. Synthesis and degradation of cyclic adenosine monophosphate (cAMP).  44 Membranes and Intracellular Signal Transduction G-Protein–Mediated Signal Desensitization to Epinephrine Transduction The epinephrine receptor (b-adrenergic receptor) undergoes The epinephrine and glucagon receptors do not affect adeny- accommodation (physiologic response reduced upon repeated late kinase directly. Instead they activate a G-protein complex stimulation) to sustained, but unchanging, concentrations of that interacts with the adenylate cyclase. G-protein–coupled epinephrine. As the Gs subunits dissociate from the receptor, receptors contain seven a-helical domains (seven-helix motif) the b-adrenergic receptor kinase phosphorylates the cytoplas- extending across the membrane. An extracellular domain con- mic domain of the receptor (see Fig. 5-8). The phosphorylated tains the hormone-binding site, and an intracellular domain in- domain will not interact with Gs protein even with epineph- teracts with the G-proteins. The hormone receptor activates rine bound to the receptor. Since the kinase phosphorylates either stimulatory or inhibitory G-proteins (Table 5-2). The only the hormone-receptor complex and not the free receptor, process for activation of adenylate cyclase (Fig. 5-8) follows: the concentration of epinephrine must increase to generate a 1. Hormone binding causes a change in intracellular domain, new active hormone-receptor complex. If epinephrine levels allowing interaction with the heterotrimeric Gs protein. remain constant, no active receptor is available, even if it 2. The a-subunit of the Gs protein releases bound guanosine di- binds epinephrine. In this way, the epinephrine sensitivity phosphate (GDP) and binds guanosine triphosphate (GTP). of the cell will decrease with constant stimulation, yielding 3. The a-subunit–GTP complex dissociates from the b-g a refractory state. dimer and interacts with adenylate cyclase. 4. Binding one hormone molecule causes the formation of many active a-subunits; this amplifies the hormonal signal. Phosphoinositide Cascade 5. The a-subunit deactivates itself within minutes by hydrolyz- Some hormones such as angiotensin II, epinephrine (a1- ing GTP to GDP (GTPase activity); the GDP remains bound. receptors), vasopressin, and oxytocin stimulate the action of 6. The a-subunit–GDP complex reassociates with the b-g dimer phospholipase C in the plasma membrane. Phospholipase C to form an inactive complex. (Note: Spontaneous GTP hydro- hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to lysis gives G-proteins an automatic deactivating mechanism.) produce two messenger molecules: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (Fig. 5-9). KEY POINTS ABOUT INTRACELLULAR SIGNAL Inositol 1,4,5-Trisphosphate TRANSDUCTION IP3 causes a rapid release of Caþþ from the endoplasmic retic- n Plasma membrane receptors have a hormone recognition do- ulum by opening Caþþ channels. Cytosolic Caþþ then binds main, one or more transmembrane domains, and an intracellular to the regulator protein, calmodulin. The Caþþ-calmodulin domain that generates the intracellular signal. complex then activates Caþþ-calmodulin–dependent protein n cAMP is generated by adenylate cyclase and stimulates phos- kinases. The Caþþ-calmodulin–complex also activates a phorylation throughout the cell by allosteric activation of protein Caþþ-ATPase pump, quickly restoring low intracellular kinase A. [Caþþ]. Calcium is a potent enzyme activator, and its access n The epinephrine and glucagon receptors act by causing the to the cytoplasm is tightly regulated. The response is normally dissociation of the Gs subunit from its parent G-protein; the Gs rapid and transient, paralleling the rate of muscle contraction. subunit then stimulates adenylate cyclase. Free [Caþþ] in the cytosol is normally around 100 nmol, n G-proteins have an automatic GTPase deactivating mechanism, whereas extracellular [Caþþ] is 10,000-fold higher. since they are active only when GTP is bound. Smooth muscle contraction is activated by Caþþ through this signaling mechanism (see Fig. 5-9). Uncomplexed Caþþ n Inactivation of the active epinephrine hormone-receptor complex also activates protein kinase C, which plays a role in platelet by phosphorylation desensitizes the receptor. activation and prostaglandin action. Diacylglycerol Diacylglycerol (DAG) increases the activity of protein kinase TABLE 5-2. Function of G-Proteins C by increasing its affinity for Caþþ. Protein kinase C regu- lates target proteins by serine and threonine phosphorylation. G-PROTEIN FUNCTION Note that both IP3 and DAG activate protein kinase C, but by different mechanisms. Gs Stimulates adenylate cyclase The phosphoinositide-related hormones activate the Gq (cAMP pathway) protein by allowing it to bind GTP. The active GTP-Gq com- Gi Inhibits adenylate cyclase plex then activates phospholipase C until its concentrations Gq Stimulates phospholipase C are reduced. Like the other G-proteins the Gq protein auto- (phosphoinositide pathway) matically inactivates itself by hydrolyzing its bound GTP. Transducin Stimulates cGMP phosphodiesterase As hormone concentrations drop, so does the renewal of cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine active Gq-GTP complex. Phosphatases degrade IP3 to inositol, monophosphate. and DAG is degraded to phosphatidic acid.  Intracellular signal transduction 45 H Hormone binding GTP G-protein- linked receptor γ β Adenylate cyclase α GDP Receptor interacts with GS protein GDP Receptor kinase H G-protein- ATP linked receptor γ β Adenylate α cyclase GTP α-Subunit binds GTP Pi and dissociates Adenylate cyclase is activated H ATP cAMP G-protein- linked receptor γ β Adenylate α cyclase GDP P Receptor kinase inactivates receptor Figure 5-8. Epinephrine receptor activation of Gs protein. Dissociation of Gs protein permits guanosine triphosphate (GTP) binding followed by guanosine diphosphatase activity. Inactive guanosine diphosphate (GDP) Gs reassociates with G-protein and binds to receptor for next binding of hormone. Phosphorylation of intracellular domain of the hormone-receptor complex “desensitizes” response to constant levels of epinephrine. ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate. Tyrosine Kinase Receptors 1. IRS-1 converts phosphatidylinositol in the plasma mem- Insulin and several other growth factors communicate their brane to PIP2. Protein kinase B is then activated by binding signal through tyrosine kinase receptors. Unlike the G-protein to PIP2. This route is used for short-term effects of insulin, receptors, the tyrosine kinase receptors span the plasma mem- such as increased glucose uptake and stimulation of glyco- brane with only one a-helix. The intracellular domain has two gen synthase activity. types of tyrosine kinase catalytic activity: 2. IRS-1 converts inactive ras (another type of G-protein) into 1. The receptors phosphorylate themselves (autophosphory- its active GTP-bound form. Ras-GTP activates mitogen- lation). activated protein (MAP) kinase, which then migrates to 2. They phosphorylate tyrosine residues on target proteins the nucleus to regulate gene expression. This route is used that may, in turn, become signals themselves. for the long-term effects of insulin such as increased gluco- kinase concentrations. Insulin Receptor The insulin signal is terminated by endocytosis of the insulin- The insulin receptor is a tetramer that is stabilized by internal receptor complex in endosomes formed from clathrin-coated disulfide bonds. Upon binding insulin to the external domain, pits on the plasma membrane. (Clathrin is a membrane protein the internal tyrosine kinase domain phosphorylates tyrosine designed to form lattices around membranous vesicles.) The in- residues on insulin receptor substrate 1 (IRS-1) to transduce sulin is digested, leaving clathrin and the receptor intact; they the insulin signal by two pathways (Fig. 5-10): then recycle to the plasma membrane.  46 Membranes and Intracellular Signal Transduction Hormone+Receptor Fatty acyl chains GTP Gq-GDP PIP2 (inactive) P -Inositol 4,5-bisphosphate GDP Pi Phospholipase C Gq-GTP (active) (active) Inositol 1,4,5-trisphosphate Diacylglycerol (IP3) (DAG) Ca++ release from Protein kinase C ER to cytosol activation Calmodulin Ca++ calmodulin MLC kinase Inactive ATP MLC kinase ADP Active Myosin light chains Myosin light chains (dephosphorylated) (phosphorylated) Relaxation Contraction of smooth muscle of smooth muscle Figure 5-9. Activation of phospholipase C and the phosphoinositide cascade. ER, endoplasmic reticulum; MLC, myosin light chain; PIP2, phosphatidylinositol 4,5-bisphosphate; GTP, guanosine triphosphate; GDP, guanosine diphosphate; ATP, adenosine triphosphate; ADP, adenosine diphosphate. receptors also phosphorylate tyrosine residues and undergo PHYSIOLOGY autophosphorylation. Similar to insulin, they activate the Oxytocin and Vasopressin MAP kinase pathway to regulate genes involved in cell Oxytocin and vasopressin act through the phosphoinositide division. pathway. Oxytocin stimulates smooth muscle contraction in the uterus and in the lactiferous ducts of the breast. Vaso- Nitric Oxide and Cyclic Guanosine pressin (antidiuretic hormone) increases the permeability of Monophosphate the renal collecting cell duct membranes to water, permitting Nitric oxide (NO) activates the cytosolic form of guanylate cy- greater reabsorption. clase, which increases the intracellular cyclic guanosine mono- phosphate (cGMP) concentration in vascular endothelial cells. Other Tyrosine Kinase Receptors cGMP relaxes smooth muscle and produces vasodilation. NO Monomeric tyrosine kinase receptors, like the epidermal is synthesized by NO synthase from arginine and O2, with re- growth factor receptor and platelet-derived growth factor ducing equivalents donated by reduced nicotinamide adenine receptor, aggregate on binding of the hormone. Their dinucleotide phosphate. The short (10-second) life span of NO  Intracellular signal transduction 47 Insulin P P PIP2 GTP Ras P P P P P P IRS-1 Protein kinase B Kinase P P P P (inactive) cascade P P P P MAP kinase (inactive) P P P MAP kinase Protein kinase B (active) (active) P Regulation of enzyme Gene activation activity, increased and increased glucose uptake enzyme synthesis by GLUT4 Figure 5-10. Insulin receptor with ras-dependent and ras-independent pathways. MAP kinase, mitogen-activated protein kinase. confines its action close to its source of synthesis. However, it Clinical Aspects of Intracellular readily crosses membranes to enter target cells. Nitric oxide Signaling also stimulates bactericidal activity in macrophages, inhibits platelet aggregation, and serves as a neurotransmitter in that Adenosine Diphosphate Ribosylation brain. of G-Proteins Several bacterial toxins catalyze the covalent attachment of adenosine diphosphate (ADP)-ribose to G-proteins: l Cholera toxin ADP-ribosylates the Gs a-subunit. Gs is per- PATHOLOGY manently activated and cannot hydrolyze GTP. This affects Ras Oncogene only intestinal mucosa; it produces excessive water and Ras is a G-protein, and GTP-ras stimulates normal cell growth electrolyte secretion (i.e., diarrhea). and differentiation. Its GTPase activity controls its action by l Pertussis toxin ADP-ribosylates the Gi a-subunit. Gi is per- spontaneously converting to its inactive GDP-ras form. This manently inactivated and cannot inhibit adenylate cyclase; GTPase activity keeps cell growth under control. However, the this produces whooping cough. oncogenic ras protein has very low GTPase activity and l Diphtheria toxin ADP-ribosylates eEF-2. This blocks poly- essentially adopts a constitutively active form. The cell peptide synthesis. responds as if high levels of growth factors were present, which leads to increased proliferation. Erectile Dysfunction The mechanism of erection of the penis involves release of NO in the corpus cavernosum as a result of sexual stimulation. The Intracellular Receptors of Lipophilic NO activates the enzyme guanylate cyclase, which results in Hormones increased levels of cGMP, producing smooth muscle relaxa- Cytosolic receptors bind lipophilic hormones, such as the ste- tion in the corpus cavernosum and allowing inflow of blood. roid hormones or retinoic acid (see Table 5-1). Cytosolic Drugs for treatment of erectile dysfunction enhance the ef- receptor-hormone complexes are transported to the nucleus, fect of NO by inhibiting phosphodiesterase type 5, which is where they regulate gene expression. Their action is much responsible for degradation of cGMP in the corpus caverno- slower than membrane receptor pathways, taking hours to sum. This results in smooth muscle relaxation and inflow of days to reach full effect. blood to the corpus cavernosum.  48 Membranes and Intracellular Signal Transduction MICROBIOLOGY n Insulin and other growth factors act through tyrosine kinase re- ceptors that undergo autophosphorylation in addition to phos- Cholera Toxin phorylation of tyrosine residues on signal proteins in the The cholera toxin is produced by Vibrio cholerae, pertussis cytoplasm. toxin is produced by Bordetella pertussis, and diphtheria toxin n NO is generated from arginine and stimulates guanylate cyclase is produced by Corynebacterium diphtheriae. to produce cGMP; this leads to relaxation of smooth muscle in blood vessels and vasodilation. n Cytosolic receptors bind lipophilic hormones and regulate gene expression in the nucleus. n Clinical manifestations of abnormal intracellular signaling include the action of bacterial toxins, unregulated cell growth, and erectile KEY POINTS ABOUT INTRACELLULAR dysfunction. SIGNAL RECEPTORS n The phosphoinositide cascade results when phospholipase C is stimulated by the Gq-GTP complex to produce diacylglycerol and IP3. Self-assessment questions can be accessed at www. StudentConsult.com. n IP3 creates a rapid release of Caþþ into the cell, forming a Caþþ- calmodulin complex that activates protein kinases. DAG activates protein kinase C.