32.3 How Do Signal-Transducing Receptors Respond to the Hormonal Message? 1023 helices upon hormone binding could reorient the two intracellular domains, giving rise to guanylyl cyclase activity. Nonreceptor Tyrosine Kinases Are Typified by pp60 src The first tyrosine kinases to be discovered were associated with viral transforming proteins. These proteins, produced by oncogenic viruses, enable the virus to trans- form animal cells, that is, to convert them to the cancerous state. A prime example is the tyrosine kinase expressed by the src gene of Rous or avian sarcoma virus. The protein product of this gene is pp60 v-src (the abbreviation refers to phosphopro- tein, 60 kD, viral origin, sarcoma-causing). The v-src gene was derived from the avian proto-oncogenic gene c-src during the original formation of the virus. The cellular proto-oncogene homolog of pp60 v-src is referred to as pp60 c-src . pp60 v-src is a 526-residue peripheral membrane protein. It undergoes two post-translational modifications: First, the amino group of the NH 2 -terminal glycine is modified by the covalent attachment of a myristoyl group (this modification is required for membrane association of the kinase; see Figure 32.20). Then Ser 17 and Tyr 416 are Binding site PKLD GCD active site PKLD apo GCD cGMP GTP ANP ANP PKLD GCD Complex ATP PKLD GCD ATP FIGURE 32.19 The rotation mechanism proposed by Misono and colleagues for transmembrane signaling by the ANP receptor. ANP binding causes a twist of the two extracellular domains, leading to rotation of the two intracellular domains and activation of guanylyl cyclase activity. PKLD is a protein-kinase-like domain and GCD is a guanylylcyclase domain. (Adapted from Misono, K., Ogawa, H., Qiu,Y.,and Ogata, C., 2005. Structural studies of the natriuretic peptide receptor: A novel hormone-induced rotation mechanism for transmembrane signal transduction. Peptides 26:957–968.) Ser 17 Tyr 416 (a) (b) Tyr 527 CO NH P P P FIGURE 32.20 (a) The soluble tyrosine kinase pp60 v-src is anchored to the plasma membrane via an N-terminal myristoyl group. (b) The structure of protein tyrosine kinase pp60 c-src , showing AMP–PNP in the active site (blue, green, red), Tyr 416 (orange), and Tyr 527 (yellow). Tyr 527 is phosphorylated (purple). 1024 Chapter 32 The Reception and Transmission of Extracellular Information phosphorylated. The phosphorylation at Tyr 416 , which increases kinase activity twofold to threefold, appears to be an autophosphorylation. On the other hand, phosphorylation at Tyr 527 is inhibitory and is catalyzed by another kinase known as CSK. The significance of nonreceptor tyrosine kinase activity to cell growth and transformation is only partially understood, but 1% of all cellular proteins (many of which are also kinases) are phosphorylated by these kinases. Soluble Guanylyl Cyclases Are Receptors for Nitric Oxide Nitric oxide, or NOи, a reactive free radical, acts as a neurotransmitter and as a sec- ond messenger, activating soluble guanylyl cyclase more than 400-fold. The cGMP thus produced also acts as a second messenger, inducing relaxation of vascular smooth muscle and mediating penile erection. As a dissolved gas, NOи is capable of rapid diffusion across membranes in the absence of any apparent carrier mecha- nism. This property makes NOи a particularly attractive second messenger because NOи generated in one cell can exert its effects quickly in many neighboring cells. NOи has a very short cellular half-life (1 to 5 seconds) and is rapidly degraded by nonenzymatic pathways. 32.4 How Are Receptor Signals Transduced? Receptor signals are transduced in one of three ways to initiate actions inside the cell: 1. Exchange of GDP for GTP by GTP-binding proteins (G proteins), which leads to generation of second messengers, including cAMP, phospholipid breakdown prod- ucts, and Ca 2ϩ . 2. Receptor-mediated activation of phosphorylation cascades that in turn trigger ac- tivation of various enzymes. This is the action of the receptor tyrosine kinases described in Section 32.3. Protein kinases and protein phosphatases acting as ef- fectors will be discussed in Section 32.5. 3. Conformation changes that open ion channels or recruit proteins into nuclear tran- scription complexes. Ion channels are discussed in Section 32.7, and the formation of nuclear transcription complexes was described in Chapter 29. GPCR Signals Are Transduced by G Proteins The signals of G-protein–coupled receptors (GPCRs) are transduced by GTP-binding proteins, known more commonly as G proteins. The large G proteins are hetero- trimers consisting of ␣- (45 to 47 kD), - (35 kD), and ␥- (7 to 9 kD) subunits. The ␣-subunit binds GDP or GTP and has an intrinsic, slow GTPase activity. The G ␣␥ complex (Figure 32.21, and see Figure 15.19) in the unactivated state has GDP at the A DEEPER LOOK Nitric Oxide, Nitroglycerin, and Alfred Nobel NOи is the active agent released by nitroglycerin (see accompany- ing figure), a powerful drug that ameliorates the symptoms of heart attacks and angina pectoris (chest pain due to coronary artery disease) by causing the dilation of coronary arteries. Nitro- glycerin is also the active agent in dynamite. Ironically, Alfred Nobel, the inventor of dynamite who also endowed the Nobel prizes, himself suffered from angina pectoris. In a letter to a friend in 1885, Nobel wrote, “It sounds like the irony of fate that I should be ordered by my doctor to take nitroglycerin internally.” CH 2 O NO 2 CH O NO 2 CH 2 O NO 2 ᮡ The structure of nitroglycerin, a potent vasodilator. Go to CengageNOW at www.cengage.com/login and click BiochemistryInteractive to learn more about the heterotrimeric G-protein complex. 32.4 How Are Receptor Signals Transduced? 1025 nucleotide site. Binding of hormone to receptor stimulates a rapid exchange of GTP for GDP on G ␣ . The binding of GTP causes G ␣ to dissociate from G ␥ and to associate with an effector protein such as adenylyl cyclase (Figure 32.22). Binding of G ␣ (GTP) ac- tivates adenylyl cyclase. The adenylyl cyclase actively synthesizes cAMP as long as G ␣ (GTP) remains bound to it. However, the intrinsic GTPase activity of G ␣ eventually hydrolyzes GTP to GDP, leading to dissociation of G ␣ (GDP) from adenylyl cyclase and reassocia- tion with the G ␥ dimer, regenerating the inactive heterotrimeric G ␣␥ complex. The hormone-activated GPCR is a guanine–nucleotide exchange factor (GEF)— promoting the exchange of GDP with GTP on the G protein—in a manner entirely similar to the interaction of EF-Ts with EF-Tu(GDP) (pages 969–970). By contrast, the G ␥ complex, which normally acts to inhibit the spontaneous release of GDP from G ␣ (in the inactivated state of the GPCR), is termed a guanine–nucleotide dissociation inhibitor (GDI). Other proteins may also behave as GEFs and GDIs; their actions are discussed in Section 32.5. Two stages of amplification occur in the G-protein–mediated hormone response. First, a single hormone-receptor complex can activate many G proteins before the hormone dissociates from the receptor. Second, and more obvious, the G ␣ -activated adenylyl cyclase synthesizes many cAMP molecules. Thus, the binding of hormone to a very small number of membrane receptors stimulates a large increase in concen- tration of cAMP within the cell. The hormone receptor, G protein, and cyclase con- stitute a complete hormone signal transduction unit. Hormone-receptor–mediated processes regulated by G proteins may be stimula- tory or inhibitory. Each hormone receptor interacts specifically with either a stimu- latory G protein, denoted G s , or an inhibitory G protein, denoted G i (Figure 32.22). Cyclic AMP Is a Second Messenger Cyclic AMP (denoted cAMP) was identified in 1956 by Earl Sutherland, who termed cAMP a second messenger, because it is the intracellular response pro- voked by binding of hormone (the first messenger) to its receptor. Since Suther- land’s discovery of cAMP, many other second messengers have been identified (Table 32.1). The concentrations of second messengers in cells are carefully reg- ulated. Synthesis or release of a second messenger is followed quickly by degra- dation or removal from the cytosol. Following its synthesis by adenylyl cyclase, cAMP is broken down to 5Ј-AMP by phosphodiesterase (Figure 32.23). FIGURE 32.21 A heterotrimeric G protein (␣—pink, —yellow, ␥—blue) docked with a  2 -adrenergic re- ceptor (green) (pdb id ϭ 2RH1 and 1GOT). + cAMP -Effector Stimulatory -receptor Inhibitory ␣ 2 -receptor ␣ 2 -Effector G protein GTP–GDP exchange GTP–GDP exchange G ␥ ␥  ␥  Adenylyl cyclase G i␣ G i␣ G s␣ G s␣ GDP GTP GTP GDP ATP ACTIVE FIGURE 32.22 Adenylyl cyclase activity is modulated by the interplay of stimulatory (G s ) and inhibitory (G i ) G proteins. Binding of hormones to  1 - and  2 -adrenergic receptors activates adenylyl cyclase via G s , whereas hormone binding to ␣ 2 -adrenergic receptors leads to the inhibition of adenylyl cyclase. Inhibition may occur by direct inhibition of cyclase activity by G i ␣ or by binding of G i ␥ to G s␣ . Test yourself on the concepts in this figure at www.cengage.com/login. 1026 Chapter 32 The Reception and Transmission of Extracellular Information Adenylyl cyclase (AC) is an integral membrane enzyme. Its catalytic domain, on the cytoplasmic face of the plasma membrane, includes two subdomains, denoted VC 1 and IIC 2 . Binding of the ␣-subunit of G s (denoted G s␣ ) activates the AC catalytic domain. Alfred Gilman, Stephen Sprang, and co-workers have determined the structure of a complex of G s␣ (with bound GTP) with the cytoplasmic domains (VC 1 and IIC 2 ) of adenylyl cyclase (Figure 32.24). The G s␣ complex binds to a cleft at one corner of the C 2 domain, and the surface of G s␣ -GTP that contacts adenylyl cyclase is the same sur- face that binds the G ␥ dimer. The catalytic site, where ATP is converted to cyclic AMP, is far removed from the bound G protein. cAMP Activates Protein Kinase A All second messengers exert their cellular effects by binding to one or more target molecules. cAMP produced by adenylyl cyclase activates a protein kinase, which is thus known as cAMP-dependent protein kinase. Protein kinase A, as this enzyme is also known, activates many other cellular proteins by phosphorylation. The activation of protein ki- nase A by cAMP and regulation of the enzyme by intrasteric control was described in detail in Chapter 15. The structure of protein kinase A has served as a paradigm for un- derstanding many related protein kinases (see Figure 15.9). Ras and Other Small GTP-Binding Proteins Are Proto-Oncogene Products GTP-binding proteins are implicated in growth control mechanisms in higher or- ganisms. Certain tumor virus genomes contain genes encoding 21-kD proteins that bind GTP and show regions of homology with other G proteins. The first of these Messenger Source Effect cAMP Adenylyl cyclase Activates protein kinases cGMP Guanylyl cyclase Activates protein kinases, regulates ion channels, regulates phosphodiesterases Ca 2ϩ Ion channels in ER and plasma membrane Activates protein kinases, activates Ca 2ϩ -modulated proteins IP 3 PLC action on PI Activates Ca 2ϩ channels DAG PLC action on PI Activates protein kinase C Phosphatidic acid Membrane component and product of PLD Activates Ca 2ϩ channels, inhibits adenylyl cyclase Ceramide PLC action on sphingomyelin Activates protein kinases Nitric oxide (NO) NO synthase Activates guanylyl cyclase, relaxes smooth muscle Cyclic ADP-ribose cADP-ribose synthase Activates Ca 2ϩ channels *IP 3 is inositol-1,4,5-trisphosphate; PI is phosphatidylinositol; DAG is diacylglycerol; PLC is phospholipase C; PLD is phospholipase D (see Figure 32.26). TABLE 32.1 Intracellular Second Messengers* Pyrophosphatase P P P 2 O HH HO OH H 2 O H 2 OH + CH 2 AdenineO O P O – – OO O P O – O P O – O ATP Adenine O HH HO OH CH 2 Adenine – O O P O – AMP O H HH O HH HH OOOH CH 2 O P O – Cyclic AMP Adenylyl cyclase Phosphodiesterase FIGURE 32.23 Cyclic AMP is synthesized by membrane- bound adenylyl cyclase and degraded by soluble phosphodiesterase. Go to CengageNOW at www.cengage.com/login and click BiochemistryInteractive to explore the structure and function of adenylyl cyclase. 32.4 How Are Receptor Signals Transduced? 1027 genes to be identified was found in rat sarcoma virus and was dubbed the ras gene. Genes implicated in tumor formation are known as oncogenes; they are often mu- tated versions of normal, noncancerous genes involved in growth regulation, so- called proto-oncogenes. The normal, cellular Ras protein is a GTP-binding protein that functions in a manner similar to that of other G proteins described previously, activating metabolic processes when GTP is bound and becoming inactive when GTP is hydrolyzed to GDP. The GTPase activity of the normal Ras p21 is very low, as is ap- propriate for a G protein that regulates long-term effects like growth and differenti- ation. A specific GTPase-activating protein (GAP) increases the GTPase activity of the Ras protein. Mutant (oncogenic) Ras proteins have severely impaired GTPase ac- tivity, which apparently causes serious alterations of cellular growth and metabolism in tumor cells. The conformations of Ras proteins (Figure 32.25) in complexes with GDP are different from the corresponding complexes with GTP analogs such as GMP–PNP (a nonhydrolyzable analog of GTP in which the -P and ␥-P are linked by N rather than by O). Two regions of the Ras structure change conformation upon GTP hydrolysis. These conformation changes mediate the interactions of Ras with other proteins, termed effectors. G Proteins Are Universal Signal Transducers A given G protein can be activated by several different hormone-receptor complexes. For example, either glucagon or epinephrine, binding to their distinctive receptor proteins, can activate the same species of G protein in liver cells. The effects are additive, and combined stimulation by glucagon and epinephrine leads to higher cytoplasmic concentrations of cAMP than activation by either hormone alone. G proteins are a universal means of signal transduction in higher organisms, ac- tivating many hormone-receptor–initiated cellular processes in addition to adenylyl cyclase. Such processes include, but are not limited to, activation of phospholipases (a) Dorsal surface ␣4 – 6 ␣3 – 5 ␣3Ј ␣1Ј – ␣2Ј Ventral surface SW II SW II SW I N 239 N 279 W 281 F 379 F 991 R 913 E 917 L 272 F 991 I 207 V 904 N 905 R 232 I 235 N 279 H 989 N 992 Q 236 L 914 Sw 1 IIC 2 G s␣ N N N N N C C C VC 1 (b) ␣3Ј ␣3 ␣3Ј ␣2Ј ␣2Ј ␣1Ј ␣3 ACTIVE FIGURE 32.24 (a) Two views of the complex of G s␣ with the VC 1 –IIC 2 catalytic domain of adenylyl cyclase and G s␣ . (b) Details of the G s␣ complex in the same orientation as the structures in (a). SW-I and SW-II are “switch regions,”whose conformations differ greatly depending on whether GTP or GDP is bound. (Courtesy of Alfred Gilman, University of Texas Southwestern Medical Center.) Test yourself on the concepts in this figure at www.cengage.com/login. (a) (b) FIGURE 32.25 The structure of Ras complexed with (a) GDP (pdb id ϭ 1LF5) and (b) GMP–PNP (pdb id ϭ 1LF0).The Ras p21–GMP–PNP complex is the active conformation of this protein. A Mg 2ϩ ion (red) is shown in both structures. 1028 Chapter 32 The Reception and Transmission of Extracellular Information C and A 2 and the opening or closing of transmembrane channels for K ϩ , Na ϩ , and Ca 2ϩ in brain, muscle, heart, and other organs (Table 32.2). G proteins are integral components of sensory pathways such as vision and olfaction. More than 100 dif- ferent GPCRs and at least 21 distinct G proteins are known. At least a dozen differ- ent G-protein effectors have been identified, including a variety of enzymes and ion channels. Specific Phospholipases Release Second Messengers A diverse array of second messengers are generated by breakdown of membrane phospholipids. Binding of certain hormones and growth factors to their respective receptors triggers a sequence of events that can lead to the activation of specific phospholipases. The action of these phospholipases on membrane lipids produces the second messengers shown in Figure 32.26. G Protein Location Stimulus Effector Effect G s G s G s G i G i /G o G q G olf Transducin (G t ) TABLE 32.2 G Proteins and Their Physiological Effects Liver Adipose tissue Kidney Heart muscle Brain neurons Smooth muscle cells in blood vessels Neuroepithelial cells in the nose Retinal rod and cone cells of the eye Epinephrine, glucagon Epinephrine, glucagon Antidiuretic hormone Acetylcholine Enkephalins, endorphins, opioids Angiotensin Odorant molecules Light Adenylyl cyclase Adenylyl cyclase Adenylyl cyclase Potassium channel Adenylyl cyclase, potassium channels, calcium channels Phospholipase C Adenylyl cyclase cGMP phosphodiesterase Glycogen breakdown Fat breakdown Conservation of water Decreased heart rate and pumping force Changes in neuron electrical activity Muscle contraction, blood pressure elevation Odorant detection Light detection (vision) C O O O P O Polar head group – O O O O C Action of phospholipases Phospholipid Unsaturated fatty acid (arachidonate) Eicosanoids and ? Phosphatidylinositol DAG Inositol phosphates Phosphatidylcholine DAG Sphingomyelin Ceramide (a) (b) PLD PLA 2 PLA 2 SMasePLC PLC PLC FIGURE 32.26 (a) The general action of phospholipase A 2 (PLA 2 ), phospholipase C (PLC), and phospholipase D (PLD). (b) The synthesis of second messengers from phospholipids by the action of phospholipases and sphingomyelinase (SMase). 32.4 How Are Receptor Signals Transduced? 1029 Inositol Phospholipid Breakdown Yields Inositol-1,4,5-Trisphosphate and Diacylglycerol Breakdown of phosphatidylinositol (PI) and its derivatives by phospholipase C produces a family of second messengers. In the best-understood pathway, successive phosphorylations of PI produce phosphatidylinositol-4-P (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP 2 ). Four isozymes of phospholipase C (de- noted ␣, , ␥, and ␦) hydrolyze PI, PIP, and PIP 2 . Hydrolysis of PIP 2 by phospholipase C yields the second messenger inositol-1,4,5-trisphosphate (IP 3 ), as well as another second messenger, diacylglycerol (DAG) (Figure 32.27). IP 3 is water soluble and dif- fuses to intracellular organelles where release of Ca 2ϩ is activated. DAG, on the other hand, is lipophilic and remains in the plasma membrane, where it activates a Ca 2ϩ - dependent protein kinase known as protein kinase C (see following discussion). HUMAN BIOCHEMISTRY Cancer, Oncogenes, and Tumor Suppressor Genes The disease state known as cancer is the uncontrolled growth and proliferation of one or more cell types in the body. Control of cell growth and division is an incredibly complex process, involving the signal-transducing proteins (and small molecules) described in this chapter and many others like them. The genes that give rise to these growth-controlling proteins are of two distinct types: 1. Oncogenes: These genes code for proteins that are capable of stimulating cell growth and division. In normal tissues and organisms, such growth-stimulating proteins are regulated so that growth is appropriately limited. However, mutations in these genes may result in loss of growth regulation, leading to uncontrolled cell proliferation and tumor development. These mutant genes are known as oncogenes because they induce the oncogenic state—cancer. The normal versions of these genes are termed proto-oncogenes; proto-oncogenes are essential for normal cell growth and differentiation. Oncogenes are domi- nant, because mutation of only one of the cell’s two copies of the gene can lead to tumor formation. Table A lists a few of the known oncogenes (more than 60 are now known). 2. Tumor suppressor genes: These genes code for proteins whose normal function is to turn off cell growth. A mutation in one of these growth-limiting g enes may result in a protein product that has lost its growth-limiting ability. Since the nor- mal products suppress tumor growth, the genes are known as tumor suppressor genes. Because both cellular copies of a tumor suppressor gene must be mutated to foil its growth-limiting action, these genes are recessive in nature. Table B presents several recognized tumor suppressor genes. Careful molecular analysis of cancerous tissue has shown that tumor development may result from mutations in several proto- oncogenes or tumor suppressor genes. The implication is that there is redundancy in cellular growth regulation. Many (if not all) tumors are either the result of interactions of two or more oncogene prod- ucts or arise from simultaneous mutations in a proto-oncogene and both copies of a tumor suppressor gene. Cells have thus evolved with overlapping growth-control mechanisms. When one is com- promised by mutation, others take over. Proto- Oncogene Neoplasm(s) Abl Chronic myelogenous leukemia ErbB-1 Squamous cell carcinoma; astrocytoma ErbB-2 (Neu) Adenocarcinoma of breast, ovary, and stomach Myc Burkitt’s lymphoma; carcinoma of lung, breast, and cervix H-Ras Carcinoma of colon, lung, and pancreas; melanoma N-Ras Carcinoma of genitourinary tract and thyroid; melanoma Ros Astrocytoma Src Carcinoma of colon Jun Several Fos Adapted from Bishop, J. M., 1991.Molecular themes in oncogenesis. Cell 64:235–248; Croce, C. M., 2008. Oncogenes and cancer. New England Journal of Medicine 358:502–511. TABLE A A Representative List of Proto-Oncogenes Implicated in Human Tumors Tumor Suppressor Gene Neoplasm(s) RB1 Retinoblastoma; osteosarcoma; carcinoma of breast, bladder, and lung p53 Astrocytoma; carcinoma of breast, colon, and lung; osteosarcoma WT1 Wilms’ tumor DCC Carcinoma of colon NF1 Neurofibromatosis type 1 FAP Carcinoma of colon MEN-1 Tumors of parathyroid, pancreas, pituitary, and adrenal cortex Adapted from Bishop, J. M., 1991. Molecular themes in oncogenesis. Cell 64:235–248, and Sherr, C. J., 2004. Principles of tumor suppression. Cell 116:235–246. TABLE B Representative Tumor Suppressor Genes Implicated in Human Tumors } 1030 Chapter 32 The Reception and Transmission of Extracellular Information Activation of Phospholipase C Is Mediated by G Proteins or by Tyrosine Kinases Phospholipase C-, C-␥, and C-␦ are all Ca 2ϩ -dependent, but the different phos- pholipase C isozymes are activated by different intracellular events. Phospholipase C- is stimulated by G proteins (Figure 32.28). On the other hand, phospholipase C-␥ is activated by receptor tyrosine kinases (Figure 32.29). The domain organiza- OH HO OH OH OH PI PI-4-P PI-4,5-P 2 I-1-P I-1,4-P 2 I-1,4,5-P 3 (IP 3 ) P HO OH OH OH P P HO OH OH P P P 1 6 5 4 32 1 6 5 4 32 1 6 5 4 32 DAG DAG DAG PLC PLC PLC FIGURE 32.27 The family of second messengers pro- duced by phosphorylation and breakdown of phos- phatidylinositol. PLC action instigates a bifurcating path- way culminating in two distinct and independent second messengers: DAG and IP 3 . ++ Polypeptide hormone Phospholipase C- PIP 2 IP 3 DAG+ Ca 2+ Receptor G q ␥  ␣ GTP GDP FIGURE 32.28 Phospholipase C- is activated specifically by G q , a GTP-binding protein, and also by Ca 2ϩ . ++ Polypeptide hormone Phospholipase C-␥ PIP 2 IP 3 DAG+ Ca 2+ Tyrosine kinase Receptor tyrosine kinase(RTK) P FIGURE 32.29 Phospholipase C-␥ is activated upon phos- phorylation by receptor tyrosine kinases and by Ca 2ϩ . 32.4 How Are Receptor Signals Transduced? 1031 tion of phospholipase C- and C-␥ is shown in Figure 32.30. The X and Y domains of phospholipase C- and C-␥ are highly homologous, and both of these domains are required for phospholipase C activation. The other domains of these isozymes confer specificity for G-protein activation or tyrosine kinase activation. Phosphatidylcholine, Sphingomyelin, and Glycosphingolipids Also Generate Second Messengers In addition to PI, other phospholipids serve as sources of second messengers. Breakdown of phosphatidylcholine by phospholipases yields a variety of second messengers, including DAG, phosphatidic acid, and prostaglandins. The action of sphingomyelinase on sphingomyelin produces ceramide, which stimulates ceramide-activated protein kinase. Similarly, gangliosides (such as ganglioside G M3 ; see Chapter 8) and their breakdown products modulate the activity of pro- tein kinases and GPCRs. Calcium Is a Second Messenger Calcium ion is an important intracellular signal. Binding of certain hormones and signal molecules to plasma membrane receptors can cause transient increases in cytoplasmic Ca 2ϩ levels, which in turn can activate a wide variety of enzymatic processes, including smooth muscle contraction, exocytosis, and glycogen metabo- lism. (Most of these activation processes depend on special Ca 2ϩ -binding proteins discussed in the following section.) Cytoplasmic [Ca 2ϩ ] can be increased in two ways (Figure 32.31). As mentioned briefly earlier, cAMP can activate the opening of plasma membrane Ca 2ϩ channels, allowing extracellular Ca 2ϩ to stream in. On the other hand, cells also contain intracellular reservoirs of Ca 2ϩ , within the endoplas- mic reticulum and calciosomes, small membrane vesicles that are similar in some ways to muscle sarcoplasmic reticulum. These special intracellular Ca 2ϩ stores are not released by cAMP. They respond to IP 3 , a second messenger derived from PI. Intracellular Calcium-Binding Proteins Mediate the Calcium Signal Given the central importance of Ca 2ϩ as an intracellular messenger, it should not be surprising that complex mechanisms exist in cells to manage and control Ca 2ϩ . When Ca 2ϩ signals are generated by cAMP, IP 3 , and other agents, these signals are translated into the desired intracellular responses by calcium-binding proteins, which in turn regulate many cellular processes (Figure 32.32). One of these, protein kinase C, is de- scribed in Section 32.5. The other important Ca 2ϩ -binding proteins can, for the most part, be divided into two groups on the basis of structure and function: (1) the calcium-modulated proteins, including calmodulin, parvalbumin, troponin C, and many others, all of which have in common a structural feature called the EF hand (Figure 32.33), and (2) the annexin proteins, a family of homologous proteins that interact with membranes and phospholipids in a Ca 2ϩ -dependent manner. PLC-␥ 1 ␥-type XYSH2 SH2 SH3 PLC- 1 -type XY PLC-␦ 1 ␦-type XY FIGURE 32.30 The amino acid sequences of phospho- lipase C isozymes , ␥, and ␦ share two homologous domains, denoted X and Y. The sequence of ␥-isozyme contains src homology domains, denoted SH2 and SH3. SH2 domains (approximately 100 residues in length) in- teract with phosphotyrosine-containing proteins (such as RTKs), whereas SH3 domains mediate interactions with Pro-rich sequences. (Adapted from Dennis, E., Rhee, S., Gillah, M., and Hannun, E., 1991. Role of phospholipases in gener- ating lipid second messengers in signal transduction. The FASEB Journal 5:2068–2077.) IP 3 cAMP Endoplasmic reticulum Calciosome Ca 2+ Ca 2+ Ca 2+ Ca 2+ ANIMATED FIGURE 32.31 Cytosolic [Ca 2ϩ ] increases occur via the opening of Ca 2ϩ channels in the membranes of calciosomes, the endoplasmic reticulum, and the plasma membrane. See this figure animated at www.cengage.com/login. HUMAN BIOCHEMISTRY PI Metabolism and the Pharmacology of Li ؉ An intriguing aspect of the phosphoinositide story is the specific ac- tion of lithium ion, Li ϩ , on several steps of PI metabolism. Lithium salts have been used in the treatment of manic-depressive illnesses for more than 30 years, but the mechanism of lithium’s therapeutic ef- fects had been unclear. Recently, however, several reactions in the phosphatidylinositol degradation pathway have been shown to be sensitive to Li ϩ ion. For example, Li ϩ is an uncompetitive inhibitor of myo-inositol monophosphatase (see Chapter 13). Li ϩ levels simi- lar to those used in treatment of manic illness thus lead to the ac- cumulation of several key intermediates. This story is far from com- plete, and many new insights into phosphoinositide metabolism and the effects of Li ϩ can be anticipated. 1032 Chapter 32 The Reception and Transmission of Extracellular Information + + + + + + + + + + + + Phospholipase C- PIP 2 DAG Inositol-1,4,5-P 3 Inositol-1,4-P 2 Phosphatidylserine Outside Inside Endoplasmic reticulum Protein kinase C Ca 2+ /CaM protein kinase Cellular responses Cellular responses Ca 2+ Ca 2+ Ca 2+ Inactive target protein Active target protein Inactive target protein Active target protein P P Polypeptide hormone Receptor G protein ␥  ␣ GTP GDP Inositol trisphosphatase 3 1 4 5 6 2 ACTIVE FIGURE 32.32 IP 3 -mediated signal transduction pathways. Increased [Ca 2ϩ ] activates protein kinases, which phosphorylate target proteins. Ca 2ϩ /CaM represents calci-calmodulin (Ca 2ϩ complexed with the regulatory protein calmodulin). Test yourself on the concepts in this figure at www.cengage.com/ login. (a) (b) (c) FIGURE 32.33 (a) Structure of uncomplexed calmodulin (pdb id ϭ 1LKJ). Calmodulin, with four Ca 2ϩ -binding domains, forms a dumbbell-shaped structure with two globular domains joined by an extended, central helix. Each globular domain juxtaposes two Ca 2ϩ -binding EF-hand domains. An intriguing feature of these EF-hand domains is their nearly identical three-dimensional structure despite a relatively low degree of sequence homol- ogy (only 25% in some cases). (b, c) Complex of calmodulin (red) with a peptide from myosin light chain kinase (blue); (b) side view; (c) top view (pdb id ϭ 1QTX).