Molecular and Cellular Signaling - Martin Beckerman

12.17 GPCRs Transduce Signals Conveyed by Odorants

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12.16 How G Proteins Regulate Ion Channels

G proteins activated in response to light absorption regulate ion channels. Light absorption stimulates the GEF activity of the GPCR, enabling it to catalyze GDP dissociation from the Ga. The next set of steps used by vertebrates differs from those employed by invertebrates, but both are fairly typical of GPCR signaling. In vertebrates, the Ga subunits act on phosphodiesterases (PDEs), but in invertebrates such as Drosophila they target phospholipase Cb. The ultimate effect of both kinds of second messenger signaling is to trigger changes in ion channel activities. Cyclic GMP is less frequently encountered as a second messenger than cAMP. One place where it has a prominent role is in phototransduction. In vertebrates, rod cells possess cGMP-gated ion channels. In the absence of stimulation by photons these channels are open resulting in membrane depolarization and transmitter release from the rod cells. Gt subunits, called transducins, are found in the retina where they couple to the phototransducer rhodopsin. When light strikes rhodopsin the transducins are activated. These subunits activate cGMP phosphodiesterases that hydrolyze cytosolic cGMP to GMP, thereby reducing its concentration. The cGMP-gated ion channels close, leading to reductions in intracellular calcium levels and shifts in the membrane potential towards more negative values (Figure 12.9b).

In Drosophila, phospholipase C acts on PIP2 to generate IP3 and DAG leading to the opening of members of the transient receptor potential (Trp) family of cation channels. A scaffolding protein called inactivation no afterpotential (InaD) helps organize the signaling events that follow Ga separation. This scaffolding protein comprises five PDZ domains. Recall from the last chapter that PDZ domains mediate several different kinds of proteinprotein interactions. In Drosoplila, InaD binds to the Trps and their regulators, serving as a platform for assembly of signaling complexes (Figure 12.9c). Similar proteins are found in vertebrate nerve terminals where they, too, help organize signaling complexes formed about ion channels.

12.17GPCRs Transduce Signals Conveyed by Odorants

Olfactory neurons situated in the nasal cavity express receptors for an enormously wide range of chemical signals. The chemical compounds that can be sensed, or odorants, number in the thousands. They include aromatic and alipathic alcohols, aldehydes, esters, ethers, and ketones; aromatic hydrocarbons; and alipathic acids, alkanes, and amines. Tiny changes in structure can be sensed and converted into different odor precepts. The olfactory receptors that function as chemical sensors in these neurons belong to a large family of evolutionarily ancient Group A GPCRs. Many of these receptors have little sequence homology to one another, especially in

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FIGURE 12.9. Phototransduction in vertebrates and invertebrates: (a) Light absorbed by the retinal chromophore results in photoisomerization and GPCR conformational changes leading to activation of heterotrimeric G proteins. (b) Vertebrate signal transduction in which transducin stimulates phosphodiesterase (PDE) activity leading to hydrolysis of cGMP and the closing of cGMP-gated ion channels.The insert depicts the 6-pass transmembrane topology of the subunits of both the cGMP-gated and Trp family ion channels. (c) Invertebrate signal transduction that targets phospholipase Cb, leading to activation of phospholipid and calcium second messengers, and assembly of signaling complexes organized by InaD scaffolding proteins.

transmembrane regions H3 through H6, a fact consistent with the role of these regions in forming the ligand-binding pocket.

Olfactory signal transduction is depicted in Figure 12.10. Cyclic AMP is now used as a second messenger in place of cGMP. Binding of an odorant activates the G protein signaling to adenylyl cyclase type III, which catalyzes the production of cAMP from ATP. The cAMP molecules bind to the cyclic nucleotide-gated (CNG) ion channels resulting in their opening. The initial signal is then amplified. Calcium ions entering the cell through the CNG channels bind and activate chloride channels so that not only does positive change enter the cell but negative change leaves as well.

The olfactory system uses a combinatorial coding scheme to distinguish between different odorants. A given olfactory neuron expresses a single type of odor receptor (OR) gene on its surface. Each OR can recognize multiple orodant ligands and each kind of odor stimulates a number of

12.18 GPCRs and Ion Channels Respond to Tastants

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FIGURE 12.10. Olfactory signal transduction: Binding of an odorant activates the G protein signaling to adenylyl cyclase type III that catalyzes the production of cAMP from ATP. The cAMP molecules bind to the cyclic nucleotide-gated (CNG) ion channels resulting in their opening. Calcium ions entering the cell through the CNG channels bind and activate chloride channels. Increased production of calcium and cAMP second messengers activate protein kinases such as protein kinase A (not shown) that phosphorylate the GPCR, leading to its desensitization.

different odorant receptors. Neurons expressing a specific OR gene are dispersed throughout one of four regions in the nasal cavity. The range of ligands recognized by different ORs contains overlaps, and a particular odor is coded by a combination, or pattern, of ORs situated on different cells. The odorant signals are sent from the nose to the olfactory bulb and from there to the piriform cortex. The organization and operation of the olfactory system is discussed in greater detail in Chapter 20.

There are two other families of olfactory receptors besides the odorant receptors. These are expressed in a second olfactory structure, the vomeronasal organ. The two families are known as the V1R family with about 35 members and the V2R family with approximately 150 members. These receptors may function as receptors for mammalian pheremones. Interestingly, V1Rs are Class A GPCRs and V2Rs are Class C GPCRs, paralleling the situation for taste where two families of receptors are found, one (T1Rs) belonging to Class A and a second larger one (T2Rs) to Class C.

12.18 GPCRs and Ion Channels Respond to Tastants

There are five taste modalities—salty, sweet, sour, bitter, and umami. Two of these, salty and sour (acidic), are sensed through interactions of salts and acids with specialized ion channels. These ion channels allow for the direct entry of H+, K+, and Na+ ions into cells localized in taste buds in the tongue. The influx of these ions triggers neurotransmitter release leading to the excitation of other sensory cells resulting in the perceptions of salty and sour. Sweet, bitter, and umami are more complex. These taste modalities are sensed through G protein-coupled receptors.

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Umami is the sensation produced by food additive monosodium glutamate (MSG). Glutamate is found in many protein-rich foods such as meat, milk products, and seafood, and is an important nutrient. Umami is sensed by a GPCR that is derived from mGluR4, a metabotropic glutamate receptor belonging to Class C GPCRs.The taste receptor differs from the neurotrans- mitter-detecting form in that it is missing 50% of the extracellular domain. This modification converts the receptor from a high affinity glutamate detector to a low affinity form suited for sensing amino acid and sweet tastants.

There are three receptors in this family of Class A GPCRs. They are designated as T1R1, T1R2, and T1R3. They form heterodimers with one another that transduce sweet (T1R2/T1R3) and umami (T1R1/T1R3) tastants.

Bitter is an exceptionally important modality since it can signal the presence of alkaloids and other potentially harmful toxins. A separate family of GPCRs transduces bitter signals. This family, consisting of 30 or more Class C receptors, is referred to as the T2Rs. The T2Rs are coexpressed with G protein alpha subunits known as gustducins (Gg). Gustducin is closely related to transducin (Gt) and is part of the pathway that conveys bitter signals within the cell. The distribution of receptors for taste differs from that for olfaction. The goal in olfaction is not only to recognize a wide range of odors but to discriminate among them as well. As noted above, each neuron expresses one type of olfaction receptor. Bitter tastes serve as warnings of potentially dangerous substances, and it is not necessary for the body to discriminate among the different sensations of bitter. Many types of bitter receptors are coexpressed in each cell thus maximizing sensitivity at the expense of specificity.

References and Further Reading

G Protein-Coupled Receptors

Baldwin JM, Schertler GFX, and Unger VM [1997]. An alpha carbon template for the transmembrane helices in the rhodopsin family of G protein-coupled receptors. J. Mol. Biol., 272: 144–164.

Bockaert J, and Pin JP [1999]. Molecular tinkering of G protein-coupled receptors: An evolutionary success. EMBO J., 18: 1723–1729.

Gether U [2000]. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr. Rev., 21: 90–113.

Gether U, and Kobilka BK [1998]. G protein-coupled receptors II: Mechanism of agonist activation. J. Biol. Chem., 273: 17979–17982.

Ji TH, Grossmann M, and Ji I [1998]. G protein-coupled receptors I: Diversity of receptor-ligand interactions. J. Biol. Chem., 273: 17299–17302.

Palczewski K, et al. [2000]. Crystal structure of rhodopsin: A G protein-coupled receptor. Science, 289: 739–745.

GPCR Regulation

Bockaert J, et al. [2003]. The magic‘ tail’ of G protein-coupled receptors: An anchorage for functional protein networks. FEBS Lett., 546: 65–72.

References and Further Reading

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Grimes ML, and Miettinen HM [2003]. Receptor tyrosine kinase and G proteincoupled receptor signaling and sorting within endosomes. J. Neurochem., 84: 905–918.

Hall RA, and Lefkowitz RJ [2002]. Regulation of G protein-coupled receptor signaling by scaffold proteins. Circ. Res., 91: 672–680.

Hall RA, Premont RT, and Lefkowitz RJ [1999]. Heptahelical receptor signaling: Beyond the G protein paradigm. J. Cell Biol., 145: 927–932.

Koenig JA, and Edwardson JM [1997]. Endocytosis and recycling of G proteincoupled receptors. Trends Pharmacol. Sci., 18: 276–287.

Lefkowitz RJ [1998]. G protein-coupled receptors III: New roles for receptor kinases and arrestins in receptor signaling and desensitization. J. Biol. Chem., 273: 18677–18680.

Lodowski DT, et al. [2003]. Keeping G proteins at bay: A complex between G protein-coupled receptor kinase 2 and Gbg. Science 300: 1256–1262.

Luttrell LM, and Lefkowitz RJ [2002]. The role of b-arrestins in the termination and transduction of G protein-coupled receptor signals. J. Cell Sci., 115: 455–465.

G Proteins and Their Effectors

Hamm HE [1998]. The many faces of G protein signaling. J. Biol. Chem., 273: 669–672.

Wedegaertner PB, Wilson PT, and Bourne HR [1995]. Lipid modifications of trimeric G proteins. J. Biol. Chem., 270: 503–506.

Adenylyl Cyclases and Nucleotide Phosphodiesterases

Beavo JA [1995]. Cyclic nucleotide phosphodiesterases: Functional implications of multiple isoforms. Physiol. Rev., 75: 725–748.

Cooper DMF, Mons N, and Karpen JW [1995]. Adenylyl cyclases and the interaction between calcium and cAMP signaling. Nature, 374: 421–424.

Houslay MD, and Milligan G [1997]. Tailoring cAMP-signaling responses through isoform multiplicity. Trends Biochem. Sci., 22: 217–224.

Taussig R, and Gilman AG [1995]. Mammalian membrane-bound adenylyl cyclases.

J. Biol. Chem., 270: 1–4.

The Somatosensory System and Nociception

Marceau F, Hess JF, and Bachvarov DR [1998]. The B1 receptors for kinins.

Pharmacol. Rev., 50: 357–386.

Negishi M, Sugimoto Y, and Ichikawa A [1995]. Molecular mechanisms of diverse actions of prostanoid receptors. Biochem. Biophys. Acta, 1259: 109–120.

Smith WL, Garavito RM, and DeWitt DL [1996]. Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2. J. Biol. Chem., 271: 33157–33160.

Phototransduction

Baylor D [1996]. How photons start vision. Proc. Natl. Acad. Sci. USA, 93: 560–565. Clapham DE, Runnels LW, and Strübing C [2001]. The Trp ion channel family.

Nature Rev. Neurosci., 2: 387–396.

Hardie RC, and Raghu P [2001]. Visual transduction in Drosophila. Nature, 413: 186–193.

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Kramer RH, and Molokanova E [2001]. Modulation of cyclic-nucleotide-gated channels and regulation of vertebrate phototransduction. J. Exp. Biol., 204: 2921–2931.

Odorants and Pheromones

Buck LB [2000]. The molecular architecture of odor and pheromone sensing in mammals. Cell, 100: 611–618.

Clyne PJ, et al. [1999]. A novel family of divergent seven-transmembrane proteins: Candidate ororant receptors in Drosophila. Neuron, 22: 327–338.

Firestein S [2001]. How the olfactory system makes sense of scents. Nature, 413: 211–218.

Malnic B, et al. [1999]. Combinatorial receptor codes for odors. Cell, 96: 713–723. Zhao HQ, et al. [1998]. Functional expression of a mammalian odorant receptor.

Science, 279: 237–242.

Tastants

Adler E, et al. [2000]. A novel family of mammalian taste receptors. Cell, 100: 693–702.

Chandrashekar J, et al. [2000]. T2Rs function as bitter taste receptors. Cell, 100: 703–711.

Chaudhari N, Landin AM, and Roper SD [2000]. A metabotropic glutamate receptor variant functions as a taste receptor. Nature Neurosci., 3: 113–119.

Lindemann B [2001]. Receptors and transduction in taste. Nature, 413: 219–225. Margolskee RF [2002]. Molecular mechanisms of bitter and sweet taste transduc-

tion. J. Biol. Chem., 277: 1–4.

Nelson G, et al. [2001]. Mammalian sweet taste receptors. Cell, 106: 381–390.

Problems

12.1The b-arrestins can function as switches that first shut down signaling through the G proteins and then turn on signaling through growth pathways. When switching to non-G protein modes of signaling, the arrestin molecule and the GPCR serve as a platform for the assembly of Src and other signaling proteins. The essential features that mediate these associations are the presence of peptide sequences that bind to SH2, SH3, and PDZ domains, either in the third intracellular loop or in the cytoplasmic COOH-terminal tail, and the Src non-receptor tyrosine kinase that can bind to the proline rich motifs of the b-arrestins by means of its SH3 domain. Construct a growth pathway leading to the nucleus that begins at the GPCR and is routed through Src that binds to the b-arrestins.

12.2One of the G protein alpha subunits, Gs, stimulates adenylyl cyclases to produce cAMP, which activates protein kinase A. In b2 adrenergic GPCR (b2AR) signaling, protein kinase A phosphorylates the GPCR, and this negative feedback loop not only desensitizes the receptor but also switches its G protein preference from Gs to Gi. What are two,

Problems 303

more conventional targets of protein kinase A signaling? Draw the routes starting from ligand-receptor binding. Using information presented in Tables 12.2 and 12.3, diagram some the routes of activation of protein kinase B and protein kinase C by the G proteins.

13

Cell Fate and Polarity

There are approximately 1014 cells in the human body. Some of these are heart cells; others are liver, kidney, nerve, or muscle cells. During development sets of identical progenitor cells undergo different cells fates. They diverge at the correct time to form different cell types along the appropriate body axes and boundaries. Mutual signaling between these cells drives many of these decisions so that each cell knows what kind of cell to become. These signals are transmitted from cell to cell, transduced across the plasma membrane, and sent on to the nucleus where they activate specific sets of developmental genes. Some genes are turned on and others are turned off in response to these signals.

A small number of signaling pathways guide embryonic development. In the first part of this chapter, four pathways of particular importance with respect to development—Notch, transforming growth factor-b (TGF-b), Wnt, and hedgehog—will be examined. These pathways regulate the programs of gene expression so that at the correct time in the right place cells with the same propensity for a particular cell fate give rise to daughters exhibiting differences in morphology and the mix of proteins being expressed.

A variety of stratagems are used to achieve the developmental goals. One of these is to lay down gradients of signaling proteins either on cell surfaces or in extracellular spaces that help determine cell fate when they activate receptors on a cell. These signaling proteins are known as morphogens. Another stratagem is to utilize hierarchical sequences of gene expression so that over time different progeny will become different kinds of cells. The focus in the first part of the chapter will be on how signals are sent from the cell surface to the nucleus through the four pathways. In the second part of the chapter, the goal will be to see how morphogen gradients and hierarchical patterns of gene expression guide cell fate decisions.

The Notch, TGF-b, Wnt, and Hedgehog signaling pathways are named either for the transmembrane receptor or for the molecules that serve as ligands for the receptors. These four pathways are highly conserved in multicellular organisms. They have been studied extensively in the fly (Drosophila), worms (C. elegans), and vertebrates. A variety of rather color-

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ful names have been given to signaling proteins belonging to these pathways. The names are usually derived from the types of developmental defects seen in Drosophila when the genes encoding the proteins suffer a mutation, usually of the loss-of-function type. For example, when the Hedgehog gene is mutated, a spiky process called denticles is seen, and hence the name hedgehog was given to the protein. In the Notch pathway, partial loss-of-function defects in the Notch receptor produced notches in the wing. Defects in the Groucho protein, a downstream-acting element that participates in several pathways, result in the production of bristles around the eye. These aberrant structures resemble the eyebrows of the well-known comedian and hence the name Groucho was given to the gene and its protein product.

13.1 Notch Signaling Mediates Cell Fate Decision

The Notch pathway mediates numerous cell fate decisions. The pathway has a central role in determining which cells become neurons and which do not during the early stages of development. This same pathway is utilized in a variety of cellular contexts to generate a broad spectrum of cell fate decisions. Depending on cellular context Notch pathway mediates patterning, terminal differentiation, mitosis, and apoptosis fates.

There are three core components of the Notch signaling pathway: ligands, receptors, and effectors functioning as transcription factors. Like the other central signaling pathways, these components are highly conserved across phyla, and corresponding members of the pathway for vertebrates, fly, and worm are listed in Table 13.1. Notch signals through a juxtacrine mechanism in which a Notch receptor expressed on the surface of one cell binds to a Delta/Serrate/Lin (DSL) ligand (Table 13.1) expressed on the surface of an adjacent cell. Notch molecules are 300-kDa transmembrane receptors. They possess large extracellular domain containing

29–36 tandem epidermal growth factor (EGF) repeats, and three cysteinerich Lin/Notch repeats (LNRs). The extracellular EGF and LNR repeats mediate DSL ligand binding and Notch activation. The intracellular domain contains an NLS, 6 Cdc10/ankyrin repeats and a PEST motif, the latter a region rich in prolinc (P), glatamic acid (E), serine (S), and threonine (T) residues.

TABLE 13.1. The Notch signaling pathway: Abbreviations—Suppressor of Hairless [Su(H)].

Ligands	Vertebrates	Drosophila	C. elegans
Ligands	Delta1, 2, Jagged1, 2	Delta, Serrate	LAG-2, Apx-1
Receptors	Notch1– 4	Notch, LIN-12	GLP-1
Transcription factors	CBF-1	Su(H)	LAG-1

13.2 How Cell Fate Decisions Are Mediated

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FIGURE 13.1. Structure of the Notch protein: Shown are the 180 kDa extracellular chain and the 120 kDa transmembrane/intracellular chain. Three cleavage sites are indicated in the figure; these are labeled by the corresponding proteolytic enzymes.

Notch is synthesized as a 300-kDa precursor molecule. This primary transcript is cleaved in two in the trans-Golgi network. The two fragments remain associated with one another during translocation and insertion in the plasma membrane resulting in the formation of a heterodimer. The heterodimer consists of an N-terminal 180-kDa molecule containing the extracellular EGFs and LNRs, and a smaller C-terminal 120-kDa molecule possessing a short extracellular segment, the transmembrane sequence and the cytosolic region (Figure 13.1).

The 120-kDa C-terminal chain is cleaved at several locations to create the Notch intracellular domain (NICD), a fragment that is released from the membrane and can move to the nucleus where it forms a complex with several other proteins. Su(H) binds the NICD and together the two proteins enter the nucleus (Figure 13.2). They act as transcription factors to active genes belonging to the enhancer of a split cluster, which acts to suppress neural development. In more detail, Notch and Su(H), more generally, CSL (CBF1, Su(H), Lag-1), work together to stimulate the transcription of genes belonging to the enhancer of split E(spl) cluster. The E(spl) gene products, in turn, inhibit transcription of a cluster of proneural genes referred to as the achaete-scute complex. Since these genes are not transcribed, cells transducing the Notch signals in response to Delta ligand binding are inhibited from adopting a neural cell fate. A positive feedback loop operates to help drive unambiguous decisions and the overall process is referred to as lateral inhibition.

13.2 How Cell Fate Decisions Are Mediated

Lateral inhibition and positive feedback mediates cell fate decisions. In the absence of Notch signaling most cells in an equivalence group will adopt the same primary fate. Notch signaling restricts the number of cells travel-