Which of the following signaling pathways would be likely to trigger the slowest cell response

Almost all aspects of neuronal function, from its maturation during development, to its growth and survival, cytoskeletal organization, gene expression, neurotransmission, and use-dependent modulation, are dependent on intracellular signaling initiated at the cell surface. The response of neurons and glia to neurotransmitters, growth factors, and other signaling molecules is determined by their complement of expressed receptors and pathways that transduce and transmit these signals to intracellular compartments; and the enzymes, ion channels, and cytoskeletal proteins that ultimately mediate the effects of the neurotransmitters. The molecules involved in signal transmission and transduction are highly represented in mammalian and invertebrate genomes. Individual neuronal responses are further determined by the concentration and localization of signal transduction components, many of which can be modified by the prior history of neuronal activity. Several primary classes of signaling systems, operating at different time courses, provide great flexibility for intercellular communication. One class comprises ligand-gated ion channels. This class of signaling provides fast transmission that is activated and deactivated within 10 ms. It forms the underlying “hard wiring” of the nervous system that makes rapid multisynaptic computations possible. A second class consists of receptor tyrosine kinases, which typically respond to growth factors and to trophic factors and produce major changes in the growth, differentiation, or survival of neurons. A third and largest class utilizes G protein-linked signals in a multistep process that slows the response from 100–300 ms to many minutes. The relatively slow speed is offset, however, by a richness in the diversity of its modulation and its inherent capacity for amplification and plasticity. The initial steps in this signaling system typically generate a second messenger inside the cell, and this second messenger then activates a number of proteins, including protein kinases that modify cellular processes. Signal transduction also modulates the level of transcription of genes, which determine the differentiated and functional state of cells.

Integrin Receptor Signaling

The integrin receptor family regulates cell adhesion, migration, invasion, and cell survival.280,281 Integrin receptors are heterodimeric molecules consisting of combinations of α and β subunits. Each combination dictates the spectrum of extracellular matrix components to which these receptors bind. Once bound to the extracellular matrix, the receptors recruit multiple proteins to the cell membrane, including cytoskeletal molecules such as paxillin and vinculin that form focal adhesions to extracellular matrix components.282 Unlike the RTK family, integrin receptors do not possess intrinsic kinase activity but rather promote signaling by facilitating the activation of kinases such as SRC or focal adhesion kinase.283 Integrins are also unique in that they participate in bidirectional signaling. This so-called “inside-out” signaling occurs when extracellular ligands such as cytokines trigger a signaling cascade that leads to a conformational change in the β subunit cytoplasmic tail of the integrin receptor that is transduced to the extracellular component of the receptor, resulting in increased affinity for portions of the extracellular matrix. Conversely, “outside-in” signaling involves binding of ligand to integrins that stimulate the activation of multiple intracellular signaling pathways.284

Integrins are expressed on cancer cells and have been shown to promote disease progression.280,283,285 Integrins are also present on stromal cells, including pericytes (which promote endothelial cell growth and proliferation and thus angiogenesis) and fibroblasts, where they influence the surrounding microenvironment and thus indirectly stimulate tumor growth and proliferation. For example, vascular cell adhesion molecule 1 is expressed on pericytes and binds to the integrin receptor α4β1, which is found on the surface of endothelial cells, resulting in pericyte recruitment to sites of vascular maturation.286 Integrin signaling also plays a role in the activation of MMP–2,287 which promotes cell invasion and has been shown to regulate cyclin D and cyclin-dependent kinase inhibitor expression, thereby controlling cell cycle progression.288 Finally, integrin receptor activation can lead to increased secretion of growth factors, which then stimulate tumor invasion through autocrine and paracrine mechanisms.287

Tumors that express integrin receptors include melanomas, glioblastomas, and breast cancers. In melanoma, the αvβ3 and α5β1 integrin receptors promote vertical growth and metastatic spread to lymph nodes.289,290 In glioblastoma, αvβ3 and αvβ5 are expressed mainly at the edge of tumors, suggesting a role in tumor invasion.291 The expression of the α6β4 and αvβ3 integrin receptors in breast cancer is associated with higher grade and tumor size,292 the development of bone metastases,293 and decreased survival.294 Clinical trials of monoclonal antibodies that target integrin receptors are underway in several cancer types. For example, etaracizumab, a humanized monoclonal antibody targeting αvβ3 integrin, is being tested in solid tumors and has shown activity in patients with metastatic melanoma.295 Cilengitide, an inhibitor of the αvβ3 and αvβ5 integrins, inhibits angiogenesis and tumor cell proliferation in preclinical studies and is being tested in a phase 3 trial in combination with temozolomide and radiation in patients with glioblastoma.287

Intracellular Signaling

Some intracellular signaling molecules involved in the brain neural networks controlling eating have been identified. These include adenosine 44/42 mitogen-activated protein kinase (MAPK or ERK1/2), cAMP response element-binding protein (CREB), signal transducer and activator of transcription 3 (STAT3), and suppressor of cytokine signaling 3 (SOCS3). In addition, the immediate early gene products c-Fos and c-Jun, which are activated by neural activity, are useful markers of brain areas activated in particular functional states. The roles of these various molecules are not yet well established. Activation (i.e., phosphorylation) of MAPK is especially interesting because it has been reported that pharmacological inhibition of MAPK affects eating, indicating that it has a necessary role under some circumstances.

6.09.3.3.3 Intracellular signaling pathways

Intracellular signaling pathways downstream from glutamatergic signaling and regulation by serotonin, which lead to communication between pre- and postsynaptic elements, leading to distinct neural patterns in the barrel cortex, are emerging (reviewed in Barnett et al., 2006a; Rebsam and Gaspar, 2006). A spontaneous mutation with absent barrel phenotype was noted in 1996 (Welker et al., 1996). These mice termed barrelless exhibited normal laminar and topographic distribution of TCAs, but the overall thalamocortical projection field was larger, much like that seen in MAOA knockout mice. The mutation was later identified as the adenylate cyclase type I (AC1) gene (Abdel-Majid, R. M. et al., 1998). While there are several members of adenylate cyclases, AC1 is exclusively neuronal and is activated by glutamate, NMDA receptor stimulation, and voltage-gated calcium channels. This membrane-bound enzyme regulates production of cAMP and protein kinase A (PKA) in response to calcium entry. Presence of AC1, both at the pre- and postsynaptic sites, has made it difficult to pinpoint whether TCA and barrel defects seen in AC1 knockout mice arise from pre- or postsynaptic effects. Other mutant mice relevant to intracellular signaling molecules include phospholipase C-β1 (PLC-β1) knockout mice, which exhibit barrel cortex defects (Hannan et al., 2001). Phospholipase C (PLC) subtypes beta, gamma, and delta are activated by cell surface receptors. These phosphodiasterases hydrolyze lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], generating inositol 1,4,5-trisphosphate (InsP3), a calcium-mobilizer from internal stores, and diacylglycerol (DAG), an activator of protein kinase C (Rebecchi and Pentyala, 2000; Rhee S.G. 2001). PLC-β1 class of phospholipases are downstream from synaptic activity, hence a role in activity-dependent refinement and patterning in the barrel cortex. SynGAP, a synaptic Ras GTPase activating protein also appears to be essential in patterning of the somatosensory thalamocortical projections. Examination of the SynGAP knockout mouse phenotype revealed that somatosensory thalamic patterning as well as TCA patterning is impaired and barrels failed to form (Barnett et al., 2006b). Collectively, all of these experimental findings reveal multifaceted role of neural activity in patterning connections within topographically organized sensory maps in the brain, in particular in the somatosensory cortex. Targeted gene mutations or experimental alterations will undoubtedly reveal many if not all the key players of developmental mechanisms underlying somatosensory cortical differentiation and patterning.

5 Conclusion

Intracellular signaling pathways contribute to the neuronal plasticity that underlies long-lasting changes in behavior resulting in nicotine addiction. As reviewed briefly here, DARPP-32 regulation is a complex interplay between the four main phosphosites on the protein and the various regulatory signals that converge on DARPP-32. Importantly, these effects are specific to cell type, brain region, duration of drug administration, and drug dose. nAChRs are widely expressed across the brain, on soma and dendrites but particularly at presynaptic sites, where they modulate the release of various neurotransmitters that can subsequently affect DARPP-32 phosphorylation at their postsynaptic targets. Better understanding of DARPP-32 regulation in nicotine addiction will depend on diving into these specificities.

Compared to DARPP-32, there is more abundant evidence linking nAChR signaling to activation of CaMKII, and relatively good evidence for the role of CaMKII in mediating behaviors relevant to nicotine addiction. However, there is a lack of information on the role CaMKII plays in different stages of nicotine addiction, especially relapse. In addition, more work needs to be done to differentiate CaMKII and nAChR interactions in postsynaptic vs. presynaptic compartments. Finally, while proteomic studies have identified direct molecular interactions between CaMKII and nAChRs, more research is needed on how CaMKII is activated upstream of nAChRs, and on the contribution of CaMKII phosphorylation of nAChRs to long-term changes following nicotine exposure. There may be regional differences in different brain areas, and nAChR subtype-specific effects in this regulatory mechanism, as has been shown for effects of CaMKII downstream of nAChRs.

Finally, it is important to note, given the dedication Dr. Greengard had to promoting women in science as exemplified by the Pearl Meister Greengard Prize and the female scientists he trained in his lab, that the effects of sex on these signaling pathways have not been investigated. It is clear that sex hormones can alter DA dynamics, and it is therefore likely that there are sex differences in the intracellular signaling pathways downstream of nicotine-dependent DA signaling (Dluzen & Anderson, 1997; Zhang, Yang, Yang, Jin, & Zhen, 2008). Sex differences in nicotine addiction-related behaviors have been observed in both clinical and preclinical studies, underlining their importance (Flores, Pipkin, Uribe, Perez, & O’Dell, 2016; Lee, Calarco, McKee, Mineur, & Picciotto, 2020; Pogun & Yararbas, 2009; Smith, Bessette, Weinberger, Sheffer, & McKee, 2016). Future studies comparing tissue from male and female animals will be needed to elucidate any potential sex differences which would be essential for the development of specialized and more effective treatments for smoking, particularly in women.

3.3.1 cAMP-Dependent Pathways

Intracellular signaling pathways are a source for drug target search not only in antiparasitic drug development but also in other fields including inflammation, neurodegeneration and cancer (Cunningham et al., 2008; Lugnier, 2006; Savai et al., 2010). In some lymphomas, inhibition of cAMP-dependent protein kinases or phosphodiesterases (PDEs) interferes in cAMP-related signaling pathways (Lerner et al., 2000). The classical approach is that the putative target enzymes are cloned from drug-susceptible parasites (genome mining), and inhibition constants are determined in the presence of the drug and derivatives (Liotta and Siekierka, 2010). The underlying idea is that like in cancer cells, these drugs interfere with the cAMP-dependent regulation of the cell cycle of the parasites, thereby inhibiting proliferation and/or differentiation (Hammarton et al., 2003).

Plasmodium contains a panel of cAMP-dependent and other protein kinases (Leroy and Doerig, 2008) which may constitute suitable targets for antimalarial drug development (Jirage et al., 2010). Obviously, similar studies have to be performed with enzymes from the (hopefully) resistant host in order to forecast potential side effects. In a recent study, the cAMP-dependent protein kinase PKA from P. falciparum has gained importance as a target since it has important differences to PKAs from mammalian hosts (Haste et al., 2012). In a study concerning the potential effects of various antimalarials on host cells, inhibition of cAMP-dependent protein kinase from rat liver and bovine heart and of calmodulin-dependent myosin light chain kinase has been studied in the presence of halofantrine and related phenantrenes. In this study, also mefloquine, allegedly acting on a completely different target (see section 4.2.) inhibits one of those signaling proteins, namely calmodulin-dependent myosin light chain kinase with high affinity (Wang et al., 1994).

Moreover, adenylyl cyclases, the enzymes that synthesize cAMP, may be valuable targets. In P. falciparum, one of the two cyclases, PfACβ, plays an important role during the erythrocytic stage of the life cycle. PfACβ is susceptible to a number of small molecule inhibitors, one of them being selective for PfACβ relative to its human ortholog, soluble adenylyl cyclase (Salazar et al., 2012).

PDEs have more recently been identified as novel drug targets that exhibit a great potential as antiparasitic agents. All protozoan parasites for which genome data are available code for at least one class1 PDE. The amino acid sequences of these enzymes are reasonably well conserved among the parasites, as well as between them and the human PDE families. Considering the current wealth of inhibitors for and information about the human PDEs, the parasite PDEs are being exploited as drug targets, with medicinal chemistry developing corresponding inhibitors, which specifically target the parasite PDEs but not the human PDEs. This approach has been followed up in more depth for two related parasites: Trypanosoma brucei (Oberholzer et al., 2007), the causative agent of African human sleeping sickness, and Leishmania major, the causative agent of cutaneous leishmaniasis (Wang et al., 2007). Both organisms, as well as all their other relatives for which genome information is available, namely Trypanosoma cruzi (Chagas disease), Leishmania infantum (visceral leishmaniasis), Leishmania braziliensis (mucocutaneous leishmaniasis) and Leishmania tarentolae (a model organisms derived from lizards) code for the same set of four PDEs (Alonso et al., 2007; Kunz et al., 2005). A typical approach for studies on PDEs is to express the gene of interest in a PDE-deficient yeast strain and to characterize it by complementation of vital functions of the deficient yeast strain and by functional assays after purification of the recombinant protein as shown for three Leishmania PDEs (Johner et al., 2006) and for a Trypanosoma PDE (Alonso et al., 2007). Leishmania PDE1 has been crystallized and structural data for the design of novel inhibitors are available (Wang et al., 2007). Thus, PDEs from Leishmania and Trypanosoma spp. are candidates for a target-based development of drugs against these parasites (Seebeck et al., 2011).

Function

Intracellular signaling: Various agonist-triggered membrane receptors use inositol phospholipid hydrolysis as the first step of intracellular cascades that continue downstream via calcium and protein kinase C-mediated signaling. The membrane receptors activate a specific zinc-containing phospholipase C (EC3.1.4.3) that releases two signaling compounds: diacyl glycerol and inositol-1,4,5-triphosphate.

Eicosanoid synthesis: Phosphatidylinositol is a significant source of arachidonic acid and other long-chain polyunsaturated fatty acids for the intracellular synthesis of prostaglandins, thromboxans, leukotrienes, and other eicosanoids. Phosphatidylinositol deacylase (phosphatidyl inositol phospholipase A2, EC3.1.1.52) releases the precursor fatty acids in a tightly regulated fashion.

GPI anchors: Many proteins, such as the prion protein presumably involved in variant Creutzfeld–Jakob disease (more commonly known as mad cow disease), are specifically anchored to membranes through a GPI extension. Such GPI-anchored exoproteins can be released from membranes by HDL-associated phosphatidylinositol glycoprotein phospholipase D (EC3.1.4.50).

Diverse inositol effects: A reduction in lung cancer risk in the postinitiation period has been observed in experimental models (Wattenberg, 1999). Evidence is limited or disputed for suggested inositol effects on sperm maturation, maintenance of microtubule function and stability, and maturation of newborn lungs. It also remains to be studied whether humans, like rodents in a preliminary study (Juriloff and Harris, 2000), have an increased risk of NTDs when they have less than optimal inositol intake. Animal experiments suggest that high inositol intake may cause neuropathy, especially with an imbalance of the polyol pathway in diabetics (Williamson et al., 1986).

Osmoregulation: Inositol is one of several compounds that can protect cells from damage due to high osmotic pressure. Tubular cells in the renal papillae, where osmotic pressure is highest, promote the uptake of inositol from the intraluminal fluid via the myo-inositol/sodium cotransporter (Burger-Kentischer et al., 1999). Brain cells also contain large amounts of inositol and increase its uptake in response to hyperosmolarity. Since the inositol transporter gene is located on chromosome 21, excessive uptake due to the 50% higher gene dose in trisomy 21 may disrupt normal neuronal function in people with Down syndrome (Berry et al., 1999).

In addition, inositol-phosphates are important regulars of water disposition through the arginine vasopressin/aquaporin-2 signaling pathway in renal collecting ducts (Pernot et al., 2011).

Mental health: One of the tissues with a very active inositol phosphate metabolism is the brain. Inositol-1,4-bisphosphate 1-phosphatase (EC3.1.3.57) and myo-inositol-1(or 4)-monophosphatase (EC3.1.3.25) are brain enzymes of particular interest because both are inhibited by lithium and may be relevant targets of lithium treatment for manic depression (Patel et al., 2002). High doses (18 g/day) of inositol intake appear to be effective for the treatment of some mental disorders, including depression, panic disorder, and obsessive-compulsive disorder (Palatnik et al., 2001).

Intracellular phytate effects: Dietary phytate has been reported to reduce carcinogenesis in various standard animal models (Shamsuddin, 1999). It may be relevant in this respect that IP6 directly up-regulates the expression of p21WAF-1/CIP1 in a dose-dependent manner.

Inhibition of intestinal cation absorption: Both phytate (IP6) and IP5 potently decrease the bioavailability of concurrently consumed nonheme iron (Sandberg et al., 1999). The minute amount of 2 mg of phytate (corresponding to 20 ml of brewed black tea) can decrease absorption by 18%, 25 mg of phytate (250 ml of tea) cuts absorption in half, and 900 mg (for instance, in 60 g of mixed nuts) reduces it to 85% (Hallberg et al., 1989; Siegenberg et al., 1991). The inhibiting effect can be counteracted by large amounts (100 mg or more) of ascorbate. Inositol derivatives with fewer phosphate groups interfere to a much lesser degree. Phytate also decreases absorption of zinc very significantly (Lönnerdal, 2000), while absorption of copper (Lönnerdal et al., 1999) is affected to a much lesser extent, and the other divalent metal cations (manganese, calcium, and magnesium) only minimally.

BIOLOGICAL ACTIONS OF SYSTEMIN FAMILY MEMBERS

Intracellular signaling in response to LeSys involves rapid early events including ion transport, the activation of MAP kinase, and phospholipase activities [9, 12, 26, 30], followed by the release of linolenic acid from chloroplast membranes and its conversion to jasmonates through the octadecanoid pathway [29]. Also involved in the defense signaling pathway are ethylene, abscisic acid, and hydrogen peroxide [6, 16, 24, 29]. LeproSys is synthesized in phloem parenchyma cells [14], while systemin signals the synthesis of jasmonic acid in nearby phloem companion cells [5, 29]. This indicates that systemin is behaving as a hormone in the classical sense.

A primary role for systemin in plant defense against herbivory was established with transgenic tomato plants that constitutively express an antisense LeproSys gene, regulated by the CaMV 35S promoter. The antisense plants do not synthesize LeproSys or LeSys and are deficient in long-distance wound signaling. The plants cannot defend themselves against attacking insects in (or by) contrast to wild-type plants [15].

Tomato plants transformed with the LeproSys gene in its sense orientation overexpressed its own gene as well as 20 signaling and defense-related “early” and “late” genes [1]. This phenotype is thought to result from the synthesis of LeproSys in cells throughout the plants where it is abnormally processed to LeSys in the absence of wounding or herbivore attacks. The plants exhibit an increased resistance toward herbivory.

The three LeHypSys peptides do not serve as systemic signals, since LeproSys antisense tomato plants are incapable of systemic signaling in response to wounding [27], but may be localized signals that amplify the jasmonate signal. LeproSys antisense plants exhibit a localized wound response, and the HypSys peptides may be part of the localized signaling.

How the combined roles of LeSys and LeHypSys are coordinated during wounding is not fully understood, but the production of multiple defense signaling peptides at wound sites may have an important role in providing an early, strong synthesis of jasmonic acid. If the enzymes that process the peptide precursors were among the proteases that are inducible by wounding, then jasmonates would induce the production of both the precursors and the peptide signals as it moves along the vascular bundles, amplifying the levels of jasmonates. Both LeproSys and LepreproHypSys are localized in phloem parenchyma cells adjacent to companion cells [5, 14, 29] where synthesis of jasmonic acid takes place, and a cooperative interaction between these cells may be important in tomato plants to achieve a strong defense response [29]. Tobacco, which has a much weaker systemic response than tomato, does not synthesize systemin but may amplify jasmonic acid synthesis by the release of the HypSys peptides as part of an amplification mechanism for jasmonates. Understanding the complex interactions between Sys, HypSys, jasmonic acid, ethylene, and H2O2, will be a major challenge for future research on wound signaling.

VIII Summary

CRH ligands activate specific intracellular signaling pathways in different cell types (see Table 2 for summary). A great deal of cross talk exists between the different CRH-R-mediated signaling pathways, in particular with regard to the cAMP/PKA pathway. The intricacies of CRH signaling probably reflect the presence of more than one receptor subtype and ligand in cells involved in orchestrating the endocrine and paracrine stress response. In addition, the coupling of these receptors to specific G-proteins provides a diversity of multiple signaling pathways that may be activated following CRH-R stimulation.

Table 2. Summary of Signaling Components Activated by CRF Ligands at CRF-Rs in Specific Tissues and Cell Types

2. Domains That Bind Phosphotyrosine-Containing Peptides

Many intracellular signaling pathways are regulated by reversible phosphorylation and/or dephosphorylation of particular tyrosine residues. These reversible modifications modulate enzymatic activities and also create or eliminate specific protein–protein interactions. Consequently, eukaryotic cells contain numerous protein domains that bind to specific phosphotyrosine-containing sequences and, in so doing, regulate signal transduction pathways (reviewed by Pawson, 1995; Cowburn, 1997). Two distinct structural classes of phosphotyrosine binding domains have been identified: the SH2 domains and the phosphotyrosine binding (PTB) domains.

It has been difficult to investigate phosphotyrosine-mediated binding interactions using DNA-encoded libraries because there is no genetic codon for phosphotyrosine. However, it has been shown that phage-displayed peptide libraries can be phosphorylated in vitro and that these modified libraries can be used to isolate phosphotyrosine-containing peptide ligands. While tyrosine kinases normally exhibit specificity for the sequences flanking the substrate tyrosine, two groups have demonstrated that prolonged exposure of phage-displayed peptide libraries to kinases results in virtually complete phosphorylation of tyrosine-containing peptides (Dante et al., 1997; Gram et al., 1997). Furthermore, these experiments showed that phosphorylation of naturally occurring tyrosines in wild-type phage coat proteins was not significant enough to interfere with subsequent selection experiments.

The phosphorylated libraries were used to investigate the binding specificities of a PTB domain (Dante et al., 1997) and an SH2 domain (Gram et al., 1997). Tyrosine-containing consensus sequences were identified in each case, and for the SH2 domain it was further demonstrated that phosphotyrosine-containing synthetic peptides corresponding to the selected sequences actually bound with greater affinity than peptides derived from the natural ligand. These results suggest that in vitro phosphorylation, and other posttranslational modifications, could further extend the utility of DNA-encoded peptide libraries.