Intracellular Signal Transduction Systems
Multicellular organisms depend on com­munication between cells to assure growth, differentiation, specific functions in different types of cells, and proper response to external stimuli. Specific cell–cell interactions between different types of cells have evolved. A common leitmotif is the specific binding of an extracellu­lar signaling molecule (ligand) to a specific re­ceptor of the target cell to trigger a specific functional response. The vast variety of molecules involved in the many different types of cells can be classified into families of related structure and function (see Lodish et al., 2000; Alberts et al., 1994). Two areas are selected here: the main intracellular functions control­ling growth and the receptor tyrosine kinases.

• Main intracellular functions controlling growth
  
  Growth factors are a large group of different ex­tracellular molecules that bind with high speci­ficity to cell surface receptors (1). Their binding to the receptor (2) activates intracellular signal transduction proteins (3). This initiates a cas­cade of events resulting in activation of other proteins (often by phosphorylation) that act as second messengers (4). Hormones of different types are a heterogeneous class of signaling molecules (5). They enter the cell either by dif­fusion through the plasma membrane or by binding to a cell surface receptor (6). Some hor­mones require an intranuclear receptor (7). Eventually the signal cascade results in activa­tion or inactivation of transcription factors (8). Before transcription and translation ensue, an elaborate system of DNA damage recognition and repair systems (9) make sure that cell pro­liferation is safe (cell cycle control, 10). In the event that faults in DNA structure have not been repaired prior to replication, an important pathway sacrifices the cell by apoptosis (cell death, 11). (Figure adapted from Lodish et al., 2000.)
  
  
• Receptor tyrosine kinase family
  
  Like the G protein-coupled receptors (GPCRs, see p. 268) and their effectors, the receptor ty­ rosine kinases (RTKs) are a major class of cell surface receptors. Their ligands are soluble or membrane-bound growth factor proteins. RTK signaling pathways involve a wide variety of other functions. Mutations in RTKs may send a proliferative signal even in the absence of a growth factor, resulting in errors in embryonic development and differentation (congenital malformation) or cancer. Of the more than twenty different RTK families, five examples are selected here: the epidermal growth factor re­ceptor (EGFR); insulin receptor (IR); fibroblast growth factor receptor (FGFR) types 1,2, and 3; platelet-derived growth factor (PDGFR); and RET (rearranged during transformation). These receptors share structural features, al­though they differ in function. All have a single transmembrane domain and an intracellular ty­rosine kinase domain of slightly varied size. The extracellular domains consist of evolutionarily conserved motifs: cystein-rich regions, im­munoglobulin (Ig)-like domains, fibronectin re­peats in the tyrosine kinase with Ig and the EGF. RTK mutations cause a group of important human diseases and malformation syndromes. The phenotypes of the mutations differ accord­ing to the particular type of RTK involved and the type of mutation.
  
  
• References
  Alberts, B., et al.: Molecular Biology of the Cell.
  3rd ed. Garland Publishing Co., New York,
  1994.
  Cohen, M.M.: Fibroblast growth factor receptor
  mutations, pp. 77–94, In: M.M. Cohen Jr.,
  R.E. MacLean, eds., Craniosynostosis, Diagnosis,
  Evaluation, and Management. 2nd ed.
  Oxford University Press, Oxford, 2000.
  Lodish, H., et al.: Molecular Cell Biology (with an
  animated CD-ROM). 4th ed.W.H. Freeman &
  Co., New York, 2000.
  Muenke, M., et al.: Fibroblast growth factor receptor–
  related skeletal disorders: craniosynostosis
  and dwarfism syndromes, pp.
  1029–1038, In: J.L. Jameson, ed., Principles
  of Molecular Medicine. Humana Press, Totowa,
  New Jersey, 1998.
  Münke, M., Schell, U.: Fibroblast-growth-factor
  receptor mutations in human skeletal disorders.
  Trends Genet. 11:308–313, 1995.
  Roberton, S. C., Tynan, J.A., Donoghue, D.J.: RTK
  mutations and human syndromes: when
  good receptors turn bad. Trends Genet.
  16:265–271, 2000.

• Cell surface receptors with direct ligand effect
  
  Many hormones cannot pass through the plasma membrane; instead, they interact with cell surface receptors. Their effects are direct and very rapid. With ligand-activated (or lig­and-gated) ion channels (1), binding of the lig­and to the receptor changes the conformation of the receptor protein. This causes an ion-specific channel in the receptor protein to open. The re­sulting flow of ions changes the electric charge of the cell membrane. Receptors with ligand­activated protein kinase (2) further activate a substrate protein. Most protein kinases phosphorylate tyrosine (tyrosine kinase), serine, or threonine by transferring a phosphate residue from adenosine triphosphate (ATP), which is then converted to adenosine di- phosphate (ADP). Other receptors mediate the removal of phosphate from a phosphorylated tyrosine side chain by means of their phosphatase activity (3). With one important type of receptor, ligand binding activates guanylate cyclase (4), which catalyzes the for­mation of cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). The cGMP functions as a second messenger and brings about a rapid change of activity of enzymes or nonenzymatic proteins. Removal or degradation of the ligand reduces the concen­tration of the second messenger and ends the reaction. (Diagrams after Lodish et al., 2000.)

• Hormones with immediate effects on cells
  
  Epinephrine, norepinephrine, and histamine act directly and very rapidly. Peptide hormones such as insulin or adrenocorticotropic hormone (ACTH) initially occur as precursor polypep­tides, which are split by specific proteases to form active molecules. Some peptide hormones are coded for by a common gene; differential RNA splicing of the transcript of this gene pro­duces different precursors for translation. (Abbreviations used: ACTH, adrenocorti­cotropic hormone; FSH, follicle-stimulating hormone; LH, leutinizing hormone; TSH, thy­roid-stimulating hormone.) (Figure data after Lodish et al., 2000.)
  
  
• Cell surface receptors with indirect ligand effect
  
  Many cell surface receptors act indirectly. When they bind to a ligand they induce a series of in­tracellular activation steps. This reaction sys­tem consists of a receptor protein, a protein (G protein) bound to a guanosine residue, and an enzyme to be activated. Ligand binding alters the receptor protein and activates the G protein (2). This moves to the effector, e.g., an enzyme complex (3), and activates it (4). In this way, a second messenger is formed that triggers further reactions in the cell, e.g., cyclic ade­nosine monophosphate (cAMP) by means of the enzyme adenylate cyclase
  
  
• References
  
Lodish, H. et al.: Molecular Cell Biology. 4th ed.
Scientific American Books, New York, 2000.
Watson, J.D., et al.: Recombinant DNA. 2nd ed.
Scientific American Books, New York, 1992.

(picture1)

• Stimulatory G protein (Gs) and hormone–receptor complex
  
  There are many endogenous messengers (hor­mones) with their own specific receptors. First the hormone binds to the receptor (formation of a hormone–receptor complex). The intra­cellular transmission of signals is mainly car­ried out by special guanine-nucleotide-binding proteins, or G proteins. By binding to guanosine triphosphate (GTP, a nucleotide composed of guanine, a sugar, and three phosphate groups), the G protein becomes activated and initiates further reactions. G proteins consist of three subunits: a, 13, andy. The a subunit (stimulatory G protein, Gs) binds to the effector protein. Im­mediately thereafter, Ga is inactivated (GTPase) by hydrolysis of GTP to GDP (guanosine di- phosphate). This transforms the G protein back into an inactive form (Gi).
  Several human diseases due to defective G pro­tein or a defective G protein receptor are known. (Clapham, 1993.)
  
  
• Four hormone classes
  Four principal classes of hormones can be differentiated: (1) amino acid derivatives such as epinephrine and epinephrine derivatives; (2) polypeptides such as glucagon; (3) steroids such as cortisol and its derivatives; and (4) fatty acid derivatives such as the prostaglandins.

  sponsible for many physiological reactions. It becomes inactivated when converted into ade­nosine monophosphate (AMP) by phos­phodiesterase. cGMP (cyclic guanosine mono- phosphate) functions in the same manner as cAMP to initiate an intracellular reaction.

• Formation and hydrolysis of cAMP
  
  The key reaction is the formation of cyclic ade­nosine monophosphate (cAMP) from adenosine triphosphate (ATP) by means of adenylate cy­clase. Intracellular cyclic AMP transmits the ac­tivation initiated by the hormone–receptor complex without a molecule having passed through the plasma membrane. cAMP is responsible for many physiological reactions. It becomes inactivated when converted into adenosine monophosphate (AMP) by phosphodiesterase. cGMP (cyclic guanosine monophosphate)
  functions in the same manner as cAMP to initiate an intracellular reaction.

• G protein cycle to activate adenylate cyclase
  
  When a hormone binds to its specific receptor, a structural change occurs (1). This activates the a subunit of the G protein, which separates from the 13 andy subunits (2). The stimulatoryG protein (Gs-a) binds to the effector protein, usu­ally adenylate cyclase, and activates it (3). cAMP is then formed from ATP, while GTP is hydro­lyzed to GDP at the G-a subunit. This inactivates the effector protein and the formation of cAMP is terminated. Thus, the signal is of very short duration, and the initial conditions are rapidly restored. Several toxins exert their activity by interrupting this cycle. For example, cholera toxin inhibits inactivation of the Gs-a protein so that adenylate cyclase remains activated and large amounts of sodium and water are lost through the intestinal mucous membranes. (Figures adapted from Watson et al., 1992).
  
  
• References
  Bourne, H.R., Sanders, D.A., McCormick, F.: The
  GTPase superfamily: conserved structure
  and molecular mechanism. Nature 349:11–
  127, 1991.
  Clapham, D.E.: Mutations in G protein-linked
  receptors: novel insights on disease. Cell
  75:1237–1239, 1993.
  Linder, M.E., Gilman, A.G.: G-Proteins, Sci. Am.
  36–43, 1992.
  Watson, J.D., et al.: Recombinant DNA. 2nd ed.
  W.H. Freeman, Scientific American Books,
  New York, 1992.

• Transmembrane structure of voltage-gated ion channels
  
  The direct flow of ions across the cell membrane is regulated by ion channels. The transmem­brane proteins, composed of several domains, are arranged so that they form pores that can be opened and closed. The simplest model is the potassium channel (1). This membrane-bound polypeptide contains six transmembrane domains. The amino and the carboxy ends of the protein lie within the cell. Changes in cell membrane potential or voltage cause the chan­nel to open (or close) in order to initiate (or ter­minate) a brief flow of ions. Domain 4, which is composed of polar amino acids, is crucial for the flow of ions. Sodium and calcium ion channels consist of four subunits (2) of similar structure, each resembling a potassium channel. The simi­larity is due to the common evolutionary origin of their genes. The four subunits of the sodium channel (3) are positioned to form a very nar­row porelike passage, much narrower than a potassium channel, through the plasma mem­brane. Ion transport is brought about by mem­brane depolarization
  
  
• Seven-helix structure of transmembrane signal transmitters
  
  Indirect transmission of signals is more frequent than the direct transport of ions or li­gand-gated impulse transmission. Here, the transmembrane protein is involved only in the first step of signal transmission. Further steps
  follow. An especially common structural motif is a transmembrane protein containing seven a helices within the plasma membrane. The amino end is extracellular; the carboxy end is intracellular. Different oligosaccharide side chains are usually bound to the extracellular domains. The intracellular domains have bind­ing sites for other molecules involved in signal transmission. The seven-helix motif is the characteristic structure of G protein-binding re­ceptors (p. 268). As the G proteins themselves, these receptors and their genes form a large family with a long evolutionary history. In yeast, they serve to discern the pheromones of the mating types (p. 186); in higher organisms they are the basis for transmitting signals of vi­sion, smell, and taste (p. 278–286). (Figure re­drawn from Stryer, 1995).
  
  
• A receptor with two transmembrane protein chains, ! and "
  
  The receptor for "-aminobutyric acid (GABA) utilizes two transmembrane protein subunits, ! and #. Both the amino and the carboxy ends are extracellular. The two chains are coded for by different genes. Several oligosaccharide side chains are present on the extracellular side. The # chain contains a phosphorylation site for cAMP-dependent protein kinase.
  
  
• References
  
  Sabatini, D.D., Adesnik, M.B.: The biogenesis of
  membranes and organelles. pp. 459–553,
  In: Scriver, C.R., et al., eds., The Metabolic
  and Molecular Bases of Inherited Disease.
  7th ed. McGraw-Hill, New York, 1995.
  Stryer, L.: Biochemistry, 4th ed. Freeman Publ.,
  San Francisco, 1995.
  Watson, J.D. et al.: Recombinant DNA, 2nd ed.,
  Scientific American Books, New York, 1992.
  
  (Picture)

• Acetylcholine as a neurotransmitter
  
  Cholinergic synapses convey the nerve impulse from one nerve cell to another or from a nerve cell to a muscle cell (motor endplate). Acetylcholine leads to postsynaptic depolarization through the release of potassium ions (K+) and the uptake of sodium ions (Na+). This process is regulated by an acetylcholine receptor.
  
  
• Acetylcholine receptors
  The acetylcholine receptors are of two genetically and functionally different types. Pharmacologically they can be differentiated according to the effects of nicotine and muscarine. The nicotine-sensitive acetylcholine receptor is an ion channel for potassium and sodium. It consists of five subunits: two !, one ", one #, and one $ (1). Acetylcholine binds as a ligand to the two ! subunits. Each subunit consists of
  four transmembrane domains (2). Each subunit is encoded by its own gene (3). These genes have similar structures and nucleotide base sequences. The ligand-gated ion channel is an example of direct transport without an intermediate carrier. A mutation in the second transmembrane region has been shown to change the ion selectivity from cations to anions (Galzi, 1992.)
  
  The muscarine-sensitive type of acetylcholine receptor is a protein that contians seven transmembrane
  subunits (4). Since each exists in the form of an ! helix, it is referred to as a sevenhelix transmembrane protein (p. 270). The amino end (NH2) lies extracellularly; the carboxy end (COOH), intracellularly. The transmembrane domains are connected by intracellular and extracellular polypeptide loops (4). Different domains of the whole protein are distinguished (5) according to location and the relative proportion of hydrophilic and hydrophobic amino acids. The amino end and the carboxy end each form a domain just like the intracellular (a–c), and extracellular portions (d–f). The transmembrane domains located within
  the plasma membrane (1–7) consist for the most part of hydrophobic amino acids. The structure of the gene product corresponds to the general structure of the gene (6). The different domains are coded for by individual exons. The DNA nucleotide sequences within functionally similar domains are similar. The seven-helix transmembrane motif occurs in many receptors. The general structures of the genes and of the gene products are very similar, but they differ in their specificity of binding to other functionally relevant molecules (G proteins). They play a role not only as neurotransmitters but also in the transmission of light, odors, and taste. (Figures based on Watson et al., 1992.)
  
  
• References
  Galzi, G.L.: Mutations in the channel domain of
  a neuronal nicotinic receptor convert ion
  selectivity from cationic to anionic. Nature
  359:500–505, 1992.
  Watson, J.D., Gilman, M., Witkowski, J., Zoller,
  M.: Recombinant DNA. 2nd ed. W.H.
  Freeman, Scientific American Books, New
  York, 1992.
  
  (picture)

• Long-QT syndrome, a genetic cardiac arrhythmia
  
  Congenital long-QT syndrome is characterized by a prolonged QT interval in the electrocardiogram (more than 460 ms, corrected for heart rate), sudden attacks of missed heart beats (syncopes) or series of rapid heart beats (torsades de pointes), and an increased risk for sudden death from ventricular fibrillation in children and young adults.
  
  
• Different molecular types of long-QT syndrome
  
  Prolongation of the QT interval in the electrocardiogram results from an increase in the duration of the cardiac action potential (1). The normal potential lasts about 300 ms (phases 1 and 2). The resting membrane potential (phase 3) is reached by progressive inactivation of calcium currents and increasing depletion of potassium currents, which repolarize the cell. In phase 0 the cell is quickly depolarized by activated sodium currents following an excitatory stimulus. LQT1 accounts for about half of the patients
  with long-QT syndrome. The gene for LQT2 encodes a 1195-amino-acid transmembrane protein responsible for the other major potassium channel that participates in phase 3 repolarization (HERG stands for (human-ether-r-go-gorelated gene, a Drosophila homologue). LQT3, a sodium channel protein, consists of four subunits, each containing six transmembrane domains and a number of phosphate-binding
  sites. Homozygosity for LQT1 (KVLQT1 gene) or LQT5 (KCNE1 gene) causes a form of long-QT syndrome associated with deafness, the Jervell and Lange-Nielsen syndrome. (Figure adapted from Ackerman and Clapham, 1997.) It is important to distinguish the different types because the choice of medication differs.
  
  
• References
  
  Ackerman, M.J., Clapham, D.D.: Ion channels—
  Basic science and clinical disease. New Eng.
  J. Med. 336: 1575–1586, 1997.
  Keating, M.T., Sanguinetti, M.C.: Molecular
  genetic insights into cardiovascular disease.
  Science 272:681–685, 1996.
  Schulze-Bahr, E., et al.: KCNE1 mutations cause
  Jervell and Lange-Nielsen syndrome. Nature
  Genet. 17:267–268, 1997.
  Schulze-Bahr, E., et al.: The long-QT syndrome.
  Current status of molecular mechanisms. Z.
  Kardiol. 88:245–254, 1999.
  Viskin, S. : Long QT syndromes and torsades de
  pointes. Lancet 354:1625–1633, 1999.
  
  

  Examples of Diseases due to Genetic Ion Channel Defects
  Disease	Inheritance	Ion Channel Gene	Locus
  Cystic fibrosis	AR	CFTR (chloride)
  Long-QT syndrome (6 types)	AD	KVLQT1 (cardiac potassium) 11p15.5 HERG 7q35–36 SCNA5 (cardiac sodium) 3p21
  Malignant hyperthermia	AD	(Ryanodine, muscle calcium) 19q13.1 (skeletal-muscle sodium) 17q23–25

  
  (Picture)

• Cystic fibrosis: clinical aspects
  
  The disease primarily affects the bronchial system and the gastrointestinal tract. Viscous mucus formation leading to frequent, recurrent bronchopulmonic infections and eventually chronic oxygen deficiency characterize the common, severe form of the disease. The average life expectancy in typical CF is about 30 years. The diseasemay take a less severe, almost mild course. Congenital bilateral absence of the vas deferens (CBAVD) occurs in 95% of patients with CF. It may be the only manifestation in individuals with different mutant allelic combinations at the CF locus.
  
  
• Positional cloning of the gene for cystic fibrosis (CF)
  
  The CFTR gene was isolated on the basis of its chromosomal location (positional cloning) on the long arm of chromosome 7 at q31 (7q31). Since the gene could be mapped to the long arm of chromosome 7 near a marker locus D7S15, a long-range restriction map comprising about 1500 kb containing the presumptive CF locus flanked by two marker loci, MET and D7S8, was constructed. From there a region of 250 kb was isolated by a combination of chromosome walking and chromosome jumping. Several genes were located in this region (candidate genes) between the marker loci D7S340 and D7S424. The gene sought was identified by the finding of mutations of this gene in patients but not in controls, by comparing with similar genes in other organisms (evolutionary conservation), by determining its exon/intron structure, by sequencing it, and by determining the expression pattern of the gene in different tissues.
  
  
• The CFTR gene and its protein
  
  The CFTR gene is large, extending over 250 kb of genomic DNA, and is organized into 27 exons (24 are shown in the diagram) encoding a 6.5 kb transcript with several alternatively spliced forms of mRNA. The CFTR protein has 1480 amino acids. It is a membrane-bound chloride ion channel regulator with several functional domains: two nucleotide-binding domains (encoded by exons 9–12 and 19–23), a regulatory domain (exons 12–14a), and two transmembrane- spanning domains (exons 3–7 and 14b– 18). Each of the two transmembrane regions consists of six transmembrane segments. The nucleotide-binding domain 1 (NBD1) confers cAMP-regulated chloride channel activity. The most common mutation (occurring in 66% of patients), a deletion of a phenylalanine codon in position 508 (!F508), is located here. The protein is a member of the ATP-binding cassette (ABC) family of transporters. The R domain contains putative sites for protein kinase A and protein kinase C phosphorylation. CFTR is widely expressed in epithelial cells. The more than 800 different mutations observed in the CFTR gene (see http://www.genet.sickkids.on.ca/cftr) can be grouped into five different functional classes: (i) abolished synthesis of full-length protein, (ii) block in protein processing, (iii) reduced chloride channel regulation, (iv) reduced chloride channel conductance, (v) reduced amount of normal CFTR protein. The underlying genetic defects include missense mutations, nonsense mutations, RNA splicing mutations, and deletions. Aside from !F508 in about 66% of patients, the most frequent mutationsworldwide are G542X (2.4%), G551D (1.6%), N1303K (1.3%), and W1282X (1.2%).
  
  
• References
  Chillon, M., et al.: Mutations in the cystic fibrosis
  gene in patients with congenital absence
  of the vas deferens. N. Engl. J. Med.
  332:1475–1480, 1995.
  Rosenbluth, D.B., Brody, S. L.: Cystic fibrosis, pp.
  329–338, In: J.L. Jameson, ed., Principles of
  Molecular Medicine. Human Press, Totowa,
  N.J., 1998.
  Rosenstein, B.J., Zeitline, P.C.: Cystic fibrosis.
  Lancet 351:277–282, 1998.
  Tsui, L.C.: The spectrum of cystic fibrosis mutations.
  Trends Genet. 8:392–398, 1992.

• Rod cells
  
  A rod cell consists of an outer segment with a photoreceptor region and an inner segment comprising cell nucleus and cytoplasm with endoplasmic reticulum, Golgi apparatus, and mitochondria. The outer segment contains about 1000 disks with rhodopsin molecules in the membrane. In the periphery, the approximately 16nm thick disks are folded by the protein peripherin. (Diagram after Stryer, 1995).
  
  
• Photoactivation
  
  In 1958, George Wald and co-workers discovered that light isomerizes 11-cis-retinal (1) very rapidly into all-trans-retinal (2), a form that practically does not exist in the dark (!1 molecule/1000 years). The light-induced structural change is so great that the resulting atomic motion can trigger a reliable and reproducible nerve impulse. The absorption spectrum of rhodopsin (3) corresponds to the spectrum of sunlight, with an optimum at a wavelength of 500 nm. Although vertebrates, arthropods, and mollusks have anatomically quite different types of eyes, all three phyla use 11-cis-retinal for photoactivation.
  
  
• Light cascade
  
  Photoactivated rhodopsin triggers a series of enzymatic steps (light cascade). First, a signaltransmitting protein of visualization, transducin, is activated by photoactivated rhodopsin. Transducin belongs to the G protein family, i.e., it can assume an inactive GDP and an active GTP form. GTP activates phosphodiesterase. This very rapidly hydrolyzes cGMP and lowers the cGMP concentration in cytosol, which leads to closure of the sodium ion channels. Immediately thereafter, phosphodiesterase is inactivated by means of a G protein cycle.
  
  
• Rhodopsin
  
  Rhodopsin is a seven-helix transmembrane protein with binding sites for functionally important molecules such as transducin, rhodopsin kinase, and arrestin on the cytosol side. The binding site of the light-sensitive molecule (chromophore) is lysine in position 296 of the seventh transmembrane domain. The light-absorbing group consists of 11-cis-retinal. The amino end of rhodopsin is located in the disk interspaces, and the carboxy end on the cytosol side. About half of the molecule is contained in the seven transmembrane hydrophobic domains, one-fourth in the disk interspaces and one-fourth on the cytosol side.
  
  
• cGMP as transmitter in the vizualization process
  
  The light cascade ends with rapid hydrolysis of cGMP, the internal transmitter in visualization. This leads to rapid closure of the sodium ion channels and hyperpolarization of the membrane to initiate nerve impulse, which is transmitted as a signal to the brain.
  
  
• References
  
  Palczewski, K. et al.: Crystal structure of
  rhodopsin: A G protein-coupled receptor.
  Science 289:739–745, 2000.
  Schoenlein, R.W., et al.: The first step in vision:
  Femtosecond isomerization of rhodopsin.
  Science 254:412–415, 1991.
  Stryer, L.: Biochemistry. 4th ed. W.H. Freeman,
  New York, 1995.
  Stryer, L.: Molecular basis of visual excitation.
  Cold Spring HarborSympQuant Biol. 53:28–
  294, 1988.

• Retinitis pigmentosa
  
  The fundus of the eye shows distinct displacement of pigmentation, with irregular hyperpigmentation and depigmentation. The papilla (optic disk) shows waxy yellow discoloration. The loss of vision, especially in dim light (night blindness), proceeds from the periphery to the center at different rates depending on the form of the disease, until only a very narrow central visual field remains. (Photograph from E. Zrenner, Tübingen.)
  
  
• Point mutation in codon 23
  
  The first point mutation demonstrated in the rhodopsin gene (Dryja et al., 1990) was a transversion from cytosine to adenine in codon 23. This changed the codon CCC for proline (Pro) into CAC for histidine (His). Since the proline in position 23 occurs in more than ten related G protein receptors, it must be very important for normal function.
  
  
• Mutations in rhodopsin
  
  The gene locus for rhodopsin (RHO) in man lies on the long arm of chromosome 3 in region 2, band 1.4 (3q21.4). Dominant and autosomal recessive inherited mutations have been demonstrated in humans. Most mutations lead to the exchange of an amino acid, although deletions may also occur. Of the 348 amino acids of rhodopsin, 38 are identical (invariant) at various positions in vertebrates. More than 100 different mutations are known for autosomal dominant inherited RP. An increasing number of mutations are recognized to cause autosomal recessive RP. In addition, mutations in several other gene loci have been recognized to lead to retinitis pigmentosa, e. g. mutations in the gene for peripherin on the short arm of chromosome 6 in humans (6p) and a locus in the centromeric region of chromosome 8. Other photoreceptor gene disease loci are the ! and " subunits of phosphodiesterase (PDE).
  
  
• Demonstration of a mutation in codon 23 by means of oligonucleotides after PCR
  
  This pedigree (1) with autosomal dominant inherited retinitis pigmentosa due to mutation in codon 23 includes 13 affected individuals in three generations (affected females, black circles; affected males, black squares). Using polymerase chain reaction (PCR) (see p. 166), Dryja et al. (1990) demonstrated the mutation in amplified fragments of exon 1 (2). The normal oligonucleotide corresponds to the normal sequence between codons 26 and 20. The mutant sequence of the oligomere RP contains the mutant sequence CAC. All affected individuals gave a hybridization signal with the RP oligomer (2) (II-2, II-12, and III-4 were not examined), whereas unaffected individuals did not (see p. 408 for demonstration of a point mutation with oligonucleotides).
  
  
• References
  
  Barkur, S. S. : Retinitis pigmentosa and related
  disorders. Am J Med Genet. 52:467–474,
  1994.
  Dryja, T.P.: Retinitis pigmentosa, pp. 4297–
  4309, In: C.R. Scriver, et al., eds., The Metabolic
  and Molecular Bases of Inherited Disease.
  7th ed. McGraw-Hill, New York, 1995.
  Dryja, T.P., et al.: A point mutation of the
  rhodopsin gene in one form of retinitis pigmentosa.
  Nature 343:364–366, 1990.
  McInnes, R.R., Bascom, R.A.: Retinal genetics: a
  nullifying effect for rhodopsin. Nature
  Genetics 1:155–157, 1992.
  Wright, A.F.: New insights into genetic eye disease.
  Trends Genet. 8:85–91, 1992.

• Genes for photoreceptor proteins in cones
  
  The gene for the blue receptor is autosomal; the genes for the red and green receptors are X chromosomal. The absorption spectra of the three receptors show maxima of 426 nm for blue, about 530 for green, and about 550 for red. The red receptorwas discovered to be polymorphic, with two somewhat different absorption maxima at 552 and 557 nm.
  
  
• Evolution of the genes for visual pigment photoreceptors
  
  The photoreceptor genes arose froma single ancestral gene (protogene). The rhodopsin–transducin pair is found in invertebrates and is at least 700 million years old. The blue receptor is almost as old as rhodopsin, about 500 million years. The separation into a receptor for green and one for red must have occurred only about 30 million years ago, after the Old World and New World apes separated, since man and the Old World apes have three cone pigments whereas NewWorld apes have two.
  
  
• Structural similarity of the visual pigments
  
  In 1986, J. Nathans and co-workers sequenced the genes for color photoreceptors and observed marked structural similarities, especially of the green and red receptor genes. Here the gene products (the receptors) are shown and their similarities are compared. The dark dots indicate variant amino acids; the light dots are identical amino acids, given in percentages.
  
  
• Polymorphism in the photoreceptor for red
  
  A. G. Motulsky and co-workers (Winderickx et al., 1992) demonstrated variant codons in three regions of the red receptor gene (1). Serine was found at position 180 in 60% of the investigated males; alanine in 40%. Position 230 showed polymorphism of isoleucine (Ile) and threonine (Thr); position 233 of alanine (Ala) and serine (Ser) (2). Differences in red color perception could be demonstrated by the color-mixing test procedure of Raleigh (3).
  
  
• Normal and defective red–green vision
  
  One gene for red and one to three genes for green lie close together on the long arm of the X chromosome in humans (1). Since the sequences of these genes are very similar, unequal crossing-over is not infrequent (2). Intergenic crossing-over leads to loss (green blindness) or duplication; intragenic crossing-over leads to a hybrid gene (red blindness). Green blindness results from loss of a gene for the green receptor; red blindness, from a defective or absent red receptor. With red–green blindness, neither a normal red nor a normal green receptor is present. About 1% of all men are red– green blind and about 2% are green blind. About 8% show weakness in differentiating red from green.
  
  
• References
  
  Kohl, S. , et al.: Total colour blindness is caused
  by mutations in the gene encoding the !-
  subunit of the cone photoreceptor cGMPgatedcation
  channel.NatureGenet.19:257–
  259, 1998.
  Motulsky, A.G., Deeb, S. S.: Color vision and its
  genetic defects, pp. 4275–4295. In: C.R.
  Scriver, et al., eds., The Metabolic and
  Molecular Bases of Inherited Disease. 7th ed.
  McGraw-Hill, New York, 1995.
  Nathans, J., Thomas, D., Hogness, D.S. : Molecular
  genetics of human color vision: the
  genes encoding blue, green, and red pigments.
  Science 232:193–202, 1986.
  Neitz, M., Neitz, J.: Numbers and ratios of visual
  pigment genes for normal red-green color
  vision. Science 267:1013–1016, 1995.
  Winderickx, J., et al.: Polymorphism in red photopigment
  underlies variation in colour
  matching. Nature 356:431–433, 1992.
  Wissinger, B., Sharpe, L.T.: Newaspects of an old
  theme: The genetic basis of human color vision.
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  1998.
  Wissinger, B., et al.: Human rod monochromacy:
  linkage analysis and mapping of a
  cone photoreceptor expressed candidate
  gene on chromosome 2q11. Genomics
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• The main components of the ear
  
  The auditory system consists of the outer ear, the middle ear, and the inner ear. Sound waves are funneled through the outer ear (auricle) and transmitted through the external ear canal to the tympanic membrane, which they cause to vibrate. These vibrations are transmitted through the tympanic cavity of the middle ear by a chain of three movable bones, the malleus, the incus, and the stapes. Three major cavities form the inner ear: the vestibule, the cochlea, and the semicircular canals. The chochlea is the site where auditory signals are processed. The cochlea contains amembranous labyrinth filled with a fluid, the endolymph. The vestibular apparatus includes three semicircular canals oriented at 90! degree angles to each other. They respond to rotatory and linear acceleration. Signals received here are transmitted via the vestibular nerve, which fuses with the cochlear nerve to form the acoustic nerve. The latter transmits the information to the brain.
  
  
• The cochlea
  
  The cochlea contains the cochlear duct, which forms the organ of Corti. The organ of Corti converts sound waves in the endolymph of the cochlea into intracellular signals. These are transmitted to auditory regions of the brain. The organ of Corti contains two types of sensory cells: one row of inner hair cells and three rows of outer hair cells. The inner hair cells are pure receptor cells. Vibrations induced by sound lead to slight deflections of the stereocilia and open potassium channels at the tips of the stereocilia. The influx of potassium ions at the tips of the cilia of the hair cells (see C) causes a change in membrane potential that results in a nerve impulse, which is transmitted as an auditory signal to the auditory cortex of the brain. Potassium ions are recycled to the supporting cells and the spiral ligament into the endolymph of the scala media. The tectorial membrane amplifies the sound waves as a resonator. (Figure adapted and redrawn from Willems, 2000.)
  
  
• The outer hair cell
  
  The outer hair cells combine sensory function with the ability to elongate and contract when acoustically stimulated. The apical pole of a hair cell carries an array of about 100 cylindrical stereocilia in a V-shaped arrangement. Each stereocilium contains an actin molecule, which enables it to elongate or to contract. The tips of the stereocilia are connected by tip links. The potassium channels are formed by the KCNQ4 protein (yellow) and by connexins (red). Important for the structural integrity and dynamics of the hair cells is a cytoskeleton involving actin, myosin 7A, myosin 15, and the protein diaphanous. (Figure adapted and redrawn from Willems, 2000.)
  
  
• Chromosomal locations of human deafness genes
  
  Almost every human chromosome harbors at least one gene involved in nonsyndromic monogenic hearing loss. The diagrammatic presentation shown here is limited to nonsyndromic hearing loss.
  
  
• References
  
  Robertson N.D., Morton, R.N.D.: Beginning of a
  molecular era in hearing and deafness. Clin.
  Genet. 55:149–159, 1999.
  Steel, K.P., Bussoli, T.J.: Deafness genes. Expressions
  of surprise. Trends Genet. 15:207–
  211, 1999.
  Willems, P.J.: Genetic causes of hearing loss.
  New Engl. J. Med. 342:1101–1109, 2000.

• Olfactory nerve cells in the nasal mucous membrane
  
  The peripheral olfactory neuroepithelium of the nasal mucous membrane consists of three cell types: olfactory sensory neurons, whose axons lead to the olfactory bulb, supporting cells, and basal cells, which serve as stem cells for the formation of olfactory neurons during the individual’s entire lifespan. Each olfactory neuron is bopolar, with olfactory cilia in the lumen of the nasal mucous membrane and a projection to the olfactory bulb, the first relay station of the olfactory systemon theway to the brain.
  
  
• Odor-specific transmembrane receptors and GTP-binding protein (Gs[olf])
  
  Each receptor in the cilia of the olfactory neurons binds specifically to one odorant. Binding of the receptor activates adenylate cyclase via a specific GTP-binding protein (stimulatory G protein of the olfactory system, Gs[olf]). This opens a sodium ion channel and initiates a cascade of intracellular signals that result in a nerve signal, which is transmitted to the brain.
  
  
• Olfactory receptor protein
  
  The cloning of a large gene family from the olfactory epithelium of the rat (Buck and Axel, 1991), demonstrated that a receptor protein contains seven transmembrane regions and shows marked structural homology with rhodopsin and !-adrenergic receptors. Unlike rhodopsin, the olfactory receptor proteins contain variable amino acids, especially in the fourth and fifth transmembrane domains. The third intracellular loop between transmembrane domains V and VI is relatively short (17 Genetics and Medicine amino acids), in contrast to other receptor proteins. It is assumed that contact with the various G proteins takes place here (Buck and Axel, 1991).
  
  
• Assignment of olfactory receptor RNA to neurons
  
  A gene for the receptor of a given odorant is expressed in an individual in only a few neurons. Ngai et al. 1993 classified individual olfactory neurons in the olfactory epithelium of the catfish (Ictalurus punctatus). Only 0.5–2% of all olfactory neurons recognize a given receptor probe such as probe 202 (1) or 32 (2). Odors are distinguished in the brain according to which neurons are stimulated. The topographical position of each neuron is specific for each odorant.
  
  
• Subfamilies within the multigene family
  
  Amino acid sequences derived from partial nucleotide sequences of cDNA clones (F2–F24) (1) investigated by Buck and Axel (1991) were very variable, especially in transmembrane domains III and IV. Within subfamilies, there was homology due to conserved sequences (2). For example, F12 and F13 differ in only 4 of 44 positions (91% identical). (Figures adapted from Buck & Axel, 1991, and Ngai et al., 1993).
  
  
• References
  
  Buck, L., Axel, R.: A novel multigene family may
  encode odorant receptors: amolecular basis
  for odor recognition. Cell 65:175–187, 1991.
  Chess, A., Femon, I.l, Cedar, H., Axel, R.: Allelic
  inactivation regulates olfactory receptor
  gene expression. Cell 78:823–834, 1994.
  Ngai, J., et al.: The family of genes encoding
  odorant receptors in the channel catfish.
  Cell 72:657–666, 1993.
  Ngai, J., et al.: Coding of olfactory information:
  topography of odorant receptor expression
  in the catfish olfactory epithelium. Cell
  72:667–680, 1993.
  Parmentier,M., et al.: Expression of members of
  the putative olfactory receptor gene family
  in mammalian germ cells. Nature 355:453–
  455, 1992.

• Mammalian chemosensory epithelia
  
  The oral and nasal cavities of mammals contain three distinct chemosensory epithelia: (i) the main olfactory epithelium (MOE) containing sensory cells with odorant receptors in the nose (see previous page), (ii) the taste sensory epithelium of the taste buds of the tongue, soft palate, and epiglottis, and (iii) the vomeronasal organ (VOM, also called Jacobson’s organ), a tubular structure in the nasal septum containing sensory cells with pheromone receptors. The main olfactory bulb (MOB) relays signals from the MOE to the olfactory cortex of the brain. The accessory olfactory bulb (AOB) relays signals from the VOM to areas of the amygdala and hypothalamus.
  
  
• Mammalian chemosensory systems
  
  The receptor cells are organized in three corresponding molecular and cellular chemosensory systems. Each neuron of the main olfactory sensory system (1) expresses one of the different olfactory receptor genes and sends axons to specific glomeruli of the main olfactory bulb (mitral cells). The odorant receptor (OR) gene family comprises about 1000 members, each encoding a seven-transmembrane cyclic nucleotide- gated channel with distinct odorant specificity (G-olfactory proteins, Golf). The bitter taste sensory system (2) connects axonal projections of receptor cells in the taste sensory epithelium of the taste buds to gustatory nuclei of the brain stem. Two families of taste receptors, the TIRs (two genes) and T2Rs (50–80 genes of the gustducin class) have been described. Two families of mammalian putative pheromone receptors (V1Rs and V2Rs) are encoded by 30–50 and over 100 genes, respectively (3).
  
  
• Taste receptor gene family
  
  The two novel taste receptor gene families, T1R1 and T1R2, are expressed in distinct subsets of taste receptor cells. The figure shows an alignment of the predicted amino acid sequences of 23 different T2 receptors (T2Rs) of human (h), rat (r), and mouse (m) origin between the first (TM1) and the third (TM3) transmembrane domain. Dark blue indicates identity in at least half of the aligned sequences; light blue represents conserved substitutions; and the remainder are divergent regions. They reflect the ability to bind many structurally different ligands. The T2R genes cluster on a few chromosomes, human chromosomes 5, 7, and 12 and mouse chromosomes 6 and 15.
  
  
• Expression of many taste receptor genes in the same cell
  
  Unlike olfactory system receptor cells, individual taste receptor cells express multiple T2R receptors. Up to ten T2R probes hybridize to only a few cells, shown darkened (1). Doublelabel fluorescent in-situ hybridization shows that different receptor genes (2, T2R-3 in green and T2R-7 in red, 3) are expressed in the same taste receptor cell. The T2Rs confer high sensitivity for bitter substances at low concentrations but do not distinguish between them. (Figures adapted fromDulac, 2000, and Adler et al., 2000.)
  
  
• References
  
  Adler, E., et al.: A novel family of mammalian
  taste receptors. Cell 100:693–702, 2000.
  Buck, L.B.: The molecular architecture of odor
  and pheromone sensing in mammals. Cell
  100:611–618, 2000.
  Chandrashekar, J., et al.: T2 Rs function as bitter
  taste receptors. Cell 100:703–711, 2000.
  Dulac, C.: The physiology of taste, vintage
  2000. Cell 100:607–610, 2000.
  Malnic, B., et al.: Combinatorial receptor codes
  for odors. Cell 96:713–723, 1999.