Neurotransmitter and Hormone Receptors
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1) Transcription of DNA and translation into protein are tightly regulated in Eukaryota cells. Give an account of the key steps involved.
Gene expression is under continuous regulation. This regulation is differentiated between Eukaryotic and Prokaryotic organisms, especially due to matters of complexity. In Prokaryotic organisms the regulation of gene expression is happening only at the stage of transcription and includes one stage. On the other hand, the regulation of gene expression in Eukaryotic organisms is happening not only in transcriptional level, but also at post-transcriptional, translation and post-translation level (Latchman 2007).
The figure on the right shows the steps of gene expression that can be regulated.
In case where the regulation of gene expression happens in transcriptional level, then the levels of cytoplasmic mRNA and nuclear RNA should be paralleled. On the other hand, in case a gene is being transcribed in the same manner in all tissues, there will be a difference in the majority of tissues between the cytoplasm mRNA and RNA, due to post-transcriptional mechanisms. As a result, in order to distinguish post-transcriptional and transcriptional regulation the first approach is to see if there are changes between the RNA levels in the tissues (Latchman 2007).
Initiation of transcription
In many cases where there is an increased level of transcription, this results from a high level of transcriptional initiation mediated by RNA polymerase responsible for initiating the process. This means that in a tissue that occurs an active transcription process, the RNA polymerases will be moving beside the gene continuously, and thus increasing the rate of transcript production. In contrast, in tissues where a gene is transcribed at low levels, the initiation of transcription will not be so apparent and the level of transcripts will be significantly low (Latchman 2007).
Except of the initiation of transcription which seems to occur in the majority of the cases, there has been demonstrated that the regulation can also happen during at a post-initiation level by producing a shortened RNA than the full length one. In this case, the transcriptional control seems to happen by blocking the elongation of a newly formed transcript (Latchman 2007).
The expression of protein-coding genes is regulated also by specific DNA elements, which are located usually upstream of the initiation site of the gene. These DNA elements can influence positively or negatively the transcription of a gene. For example, DNA sequences such as insulators and silencers, suppress the gene's transcription (Latchman 2007).
The transcription factors are also important modulatory elements of transcription. The binding of specific transcription factors on the DNA, can either activate or suppress gene transcription (Latchman 2007) .
Although so far we discussed the transcriptional control of gene expression, which corresponds to the most abundant transcriptional control mechanism, there have been cases where even though the transcription rate of a gene doesn't change, changes in the synthesis of specific proteins have occurred. This control mechanism occurs between the translational and transcription control and includes splicing of the nascent mRNA, alternative splicing, and regulation of RNA stability. The splicing of the RNA is the process in which the protein coding regions are encoded by exons, after removing the intervening sequences which are named introns (Latchman 2007). The alternative splicing, includes the differentiated processing of RNAs in order to produce different RNA variants (Black 2000).
The translational control of gene expression, corresponds to mRNA regulation and is also a very important regulatory stage. In the same manner as transcriptional control, translation can be affected either positively or negatively by altering the levels of specific translation factors, and most of the times it happens at the initiation of the translation process due to differences in the 5' untranslated region of the mRNA. During translation, the 5' un-translated region contains an AUG triplet of bases which is important to be located in a favorable context. This means, that if the ribosome is incapable of recognizing this triplet, then the initiation of translation is inhibited. For example, in a situation which is called "leaky scanning", when AUG codons are located upstream, the ribosome has a difficulty to recognize them (Wickens et al. 2000).
Furthermore, except of the recognition of the AUG codon, other coding regions seem to play an important regulatory role. For example, some frequencies which are located in the open reading frame of the mRNA (ORF) seem to cause a frameshift at a specific triplet both in viral and mammalian mRNAs (Wickens et al. 2000).
Finally, another regulatory element of gene expression includes the post-translational control. This category composes of all the mechanisms that act after translation by modifying the protein levels. Such a mechanism is ubiquitination, which will have as a result the degradation of the ubiquitinated protein in the proteasome (Wang et al. 2013).
In conclusion the transcription and translation are highly regulated processes and this take place due to the complexity of Eukaryotic cells, since strict regulation usually corresponds to high complexity.
2) Discuss the role of microtubules and their motor proteins in the motion of cilia and flagella.
Microtubules are important elements for various cell movements, such as the chromosomal separation during mitosis and the motion of flagella and cilia. This movement, which happens along microtubules, is highly dependent on proteins that use energy from ATP hydrolysis in order to produce force and movement. These proteins are members of the kinesin and dynein families (Alberts et al. 2002).
Cilia and flagella are plasma membrane projections dependent on microtubule formation, and are important for the movement in various eukaryotic organisms. In bacteria, the flagella structures are different from the eukaryotic flagella (Alberts et al. 2002). The movement of these structures has been extensively studied by using animal models, especially sea urchin models which appears to be a very powerful system for flagellar motility. Furthermore, the first scientists to observe this sliding mechanisms driven by dynein across the microtubules, were Summers and Gibbons by using dark-field microscopy (Hirose & Amos 2012).
In eukaryotic organisms the flagella and cilia are highly similar structures. Firstly, flagella are mostly found in sperm and many hair-like cellular projections with a core composed of microtubules (Cooper 2000). With their rolling motion they permit the cells to swim through liquid media. The cilia, are usually shorter than flagella but their organization tends to be similar. Their beating motion is like the breast stroke of the swimmers, and the cycles of adjacent cilia are quite asynchronous, thus producing the characteristic effect that can be observed through microscope (Alberts et al. 2002). The movement of both each flagellum and cilium is produced by the bending of each ones core, which is specifically termed as axoneme. The axoneme is a structure that is composed of microtubules in conjunction with their associated proteins in a regular and specific pattern. This pattern is composed by nine doublet microtubules which are fused together to surround a pair of microtubules. This formation is also known as the "9+2" pattern and is represented in the following picture (Cooper 2000).
This type of arrangement is commonly seen in the majority of eukaryotic flagella and cilia forms. The length of these microtubules extends along with the length of the axoneme which can reach the 200 Î¼m. At specific positions along the distance of microtubules, are located accessory proteins, providing with this a way a cross-bridging between the doublet microtubules. These molecules are dynein proteins, and are located around the perimeter of the axoneme (Cooper 2000).
Dynein is a very large molecule (2000 kd), consisting of three heavy chains in conjunction with a variable number of intermediate and light polypeptides that have a weight ranging from 14 to 120 kd (Cooper 2000). The heavy chains are forming a globular domain which binds ATP and is responsible for moving along microtubules. The intermediate chains are responsible for the assembly of the dyneins. The light chains form two distinct groups, in accordance with the molecules that are related with. For example the first group is associated with the heavy chains, while the second group with the intermediate chains. In the cilia and flagella, there is also another specific dynein group, the intraflagellar transport (IFT) dynein, which is important for the association and disassociation of these organelles, and also have transport properties of either membrane proteins or even the IFT themselves (Hirose & Amos 2012).
The ciliary dyneins, are composed of a different set of arms, the outer dynein arms and a more complex set of inner dynein arms. Each one of this structures plays a different role in the ciliary movement. The outer dynein arms are responsible for the production of the majority of the force required for the ciliary movement, while the inner dyneins, are mostly providing a precise control of this movement (Hirose & Amos 2012).
Another important feature of the cilia and flagella, is that the minus ends of each microtubule are anchored in a structure which is named basal body. This is highly similar to the centriole, and is necessary for the arrangement of the axoneme microtubules. Each one of the doublets located in the outer part of the flagella or cilia, is formed by the extension of two of the microtubules of the basal's body triplets (Cooper 2000).
The movement of cilia and flagella is caused by the relative sliding of outer microtubules, driven by the activity of axonemal dynein, and the mechanism is represented in figure 3. The dynein bases attach with the A microtubules while the head of each dynein attaches with the B microtubule. With the green color is represented the nexin link, which binds the microtubules in the axoneme. When the one doublet of microtubules, bends along with the other one, the resulting movement is bending, which is the source of the beating movements of cilia and flagella (Alberts et al. 2002). Scientific evidence suggests that upon ATP hydrolysis the dyneins change their conformational state (Hirose & Amos 2012).
3) Describe the most important classes of molecules that participate in common signal transduction pathways
In signal transduction pathways, extracellular signals such as hormones or other molecules are registered by membrane receptors and the signal is being transferred inside the cell by a set of reactions. This signal transduction can be mediated by two distinct mechanisms. The first one includes the use of receptors, and other proteins including enzymes. The second one contains a set of molecules which are known as "second messengers", that regulate the intracellular signaling (Boon 2009) .
The intracellular second messengers, are signal molecules that reach their target by diffusion. They can be divided into two different groups, those with a hydrophobic character (diacyl glycerol or phosphatidyl inositol) and the hydrophilic ones. The hydrophobic messenger are located on the membrane and they can reach the proteins located on the membrane by diffusing through the plasma membrane of the cell. The hydrophilic messengers are located in the cytoplasm and their targets are located in the cytosol as well (Boon 2009).
The most important second messengers include the cAMP, cGMP, inositol phosphates, calcium ions, diacylgrlycerol and phosphatidyl inositol phosphates.
3'-5' cyclic AMP (cAMP)
Is among the most important second messengers and regulates a variety of cellular functions, such as glycolysis, muscle contraction and ion transport. The intracellular concentration of cAMP is regulated by two factors, the adenylyl cyclase and the phopshodiestarases. The former is important for the cAMP synthesis while the latter for its degradation. The activation of adenylyl cyclase is dependent to G-protein coupled receptors and involves the participation of GÎ± and GÎ²Î³ proteins. The degradation of cAMP mediated by cAMP phosphodiesterases, which are being regulated by Ca2+/calmodulin and also by phosphorylation (Boon 2009).
In the majority of the cases, the target of the high cAMP concentration is the activation of protein kinases A (PKAs). Initially, in the absence of cAMP the PKA is organized as a tetramer, with two catalytic and two catalytic subunits, and the protein kinase is inactive. When cAMP concentration is increased by adenylyl cyclase, leads to the binding of two molecules to the regulatory subunit and the tetramer dissociation and activation of PKA (Boon 2009).
In the same manner with cAMP, the 3'-5'-cGMP is extensively spread in the intracellular space. Like in the case of cAMP , the cGMP is formed by guanylyl cyclase from GTP. The cGMP can activate cGMP-dependent protein kinases, after binding to specific locations. In contrast to the PKA kinases, the activation of cGMP-dependent protein kinases is dependent to only one protein chain of these proteins. Specifically, the binding of the cGMP to the regulatory domain of the protein, activates it and permits phosphorylation of other substrates (Boon 2009).
The inositol containing phospholipids located in the plasma membrane, are the starting points for the production of many inositol messengers as a response to extracellular or even intracellular signals. The inositol phosphates are important players for the regulation of phospholipase C, which is a very important enzyme in the metabolism of phosphatide inositol. Another important function of these phosphates, is the recruitment of Ca2+ ions that are inside storage organelles, such as mitochondria. The calcium ions are among the most ubiquitous targets of second messengers, and regulate a vast array of actions (Boon 2009).
The calcium ions can have a regulatory character in many ways, according to the time, frequency and amount of release rate. This feature, explains the complexity of Ca2+ signals. Still it is not understood how oscillatory calcium signals are regulating various processes. It has been suggested that CaM kinase II participates in the formation of repetitive calcium signals. The high intracellular concentration of calcium is temporary and usually a local phenomenon. The cell contains a variety of transport systems, which can transfer the calcium ions inside the storage locations. These transfer mechanisms are Ca2+ - ATPases, which transfer calcium against the concentration gradient. Another mechanism, is the sodium-calcium exchange proteins which are mostly apparent in muscle cells (Boon 2009).
Diacylglycerol and phospatidyl inositol phosphates
The diacylglycerol is being produced by the metabolism of the membrane phosphatidyl inositol phosphate, after the effect of two distinct enzymes, the PLCÎ³ and PLCÎ². The diacylglycerol (DAG) is then responsible for activating the protein kinase C, which plays an important role in cell proliferation through phosphorylation of various substrate proteins (Boon 2009).
Except of DAG, the metabolism of phosphatidyl inositol diphosphate, produces the inositol triphosphate, which increases intracellular calcium levels by binding to specific receptors (Boon 2009).
Conclusively, signal transduction is a highly regulated mechanism, which permits the transmission of an extracellular signal inside the cell, and a subsequent cellular response. Among the most important molecules that are participating in this process, we distinguished some members of the family of the second messengers, such as calcium ions, diacyl glycerol and phosphatidyl inositol.
4) Describe the steps of neurotransmission.
The process of neurotransmission can be divided into five distinct steps. The first includes the synthesis of the neurotransmitter, the second the storage of the neurotransmitter into transport vesicles, the third the influx of calcium and exocytosis of neurotransmitter, the fourth the binding of the neurotransmitter at the postsynaptic membrane and the fifth the deactivation of the neurotransmitter.
The low weight neurotransmitters are synthesized in the cytoplasm of the cell, where enzymes act and convert them into mature neurotransmitters. These enzymes are produced in the neuronal cell body and are transferred to the presynaptic terminal via the slow axonal transfer system. When the new transmitters are synthesized in the cytoplasm, certain mechanisms need to follow for transporting them to the synaptic membrane. On the other hand, the neuropeptides which is the other category of known neurotransmitters, are produced in the neuronal cell body, and need to be transferred a long distance in order to reach the site of secretion. For this reason, they are transported from the soma of the neuron to the presynaptic terminal with the fast axonal transport (Hyman et al. 2009).
The small-molecule neurotransmitters, such as acetylcholine and amino acids, are stored into vesicles of 40-60 nm diameter. The primary characteristic of these vesicles, is that in electron micrographs they appear with a clear center area. The storage, requires specific proteins which are located in the membrane of the synaptic vesicles in the presynaptic neuron, the vesicle monoamine membrane transporters (VMAT). In contrast to the small-molecule neurotransmitters, the neuropeptides, are stored into synaptic vesicles with a larger size (90 to 250 nm). In electron micrographs, they seem to have a center relatively more dense than the synaptic vesicles of the small neurotransmitters (Purves et al. 2001).
After the influx of calcium into the pre-synaptic cytoplasm, it begins the process of neurotransmitter release. The first step of this exocytosis, includes the docking of the synaptic vesicle membrane at the active zone's plasma membrane. The second step is priming. This corresponds to an ATP dependent maturation of the synaptic vesicles that are being docked in the membrane of the active zone but can't be induced by Ca2+. The result of this maturation, is that the primed vesicles can immediately release their content after a Ca2+ influx. The third step of this process includes the fusion of the synaptic vesicle with the presynaptic membrane, which allows the exocytosis to happen. In this step, the function of the protein synaptotagmin is of high importance, since it senses the levels of Ca2+ and regulates with this way the fusion of the vesicles. The final step of this step includes the quantal release of the neurotransmitter with a mechanism of exocytosis. The exocytosis is synchronized with the influx of Ca2+ and its induction is controlled by the depolarization of the nerve terminal. After the release of neurotransmitter at the synaptic cleft, the membrane of the vesicle will be recycled with a process of endocytosis (Hyman et al. 2009).
After the exocytosis, the neurotransmitter starts to diffuse across the synaptic cleft and targets the post-synaptic neuron and its receptors which are localized on the membrane. The binding of the neurotransmitter to the postsynaptic membrane, will cause both biochemical and electrical alterations. Specifically, an excitatory signal will depolarize the membrane and thus a positive charge will pass inside the cell. This depolarization is caused by the opening of sodium channels located on the post-synaptic membrane, which permits the influx of sodium ions across the membrane. On the other hand, an inhibitory signal will hyperpolarize the cell, and thus a positive charge will flow with a direction outside of the cell. This hyperpolarization is induced by an inhibitory postsynaptic potential (IPSP) which cause the influx of chloride ions, which make the membrane potential more negative and the propagation of the action potential significantly more difficult (Hyman et al. 2009).
When a neurotransmitter finishes its function, the next step is to be removed from the synaptic cleft. When the local neurotransmitter concentration falls, the neurotransmitter unbounds from the post-synaptic receptor. After this, the neurotransmitter can be either degraded by specific enzymes, or reuptaken by high affinity receptors. The latter corresponds to the most common way of neurotransmitter removal, and includes the reincorporation of the neurotransmitter into the pre-synaptic terminal by endocytosis. This permits the neurotransmitter to be recycled inside the cell. The remaining percentage of neurotransmitters, follows the other path of removal which corresponds to the enzymatic degradation. A characteristic enzyme is acetylcholinesterase (AChE) which degrades the neutrotransmitter acetylcholine into acetate and choline. Another examples of such enzymes include catechol-o-methyltransferase (COMT) and monoamine oxidase (MAO) (Naik 2015).
5) Describe with examples the major mechanisms of action of the different types of neurotransmitter and hormone receptors.
In 1907, the physiologist Langley, introduced the aspect of receptor molecules, in order to explain specific properties of molecules on muscle and nerve cells. The neurotransmitter receptors are proteins located in the post-synaptic plasma membrane and contain an extracellular site, specific for the binding of a neurotransmitter. The neurotransmitters, have two distinct families of receptors. The first one, the ligand-gated ion channels or ionotropic receptors, combine the properties of ion channels by having also a neurotransmitter binding domain on their outer part of the membrane. The other family of receptors, are the metabotropic receptors, because the movement of ions depends on certain metabolic steps. The important difference of these channels with the ionotropic channels, is that they don't have an ion channel in their structure, in contrast they have a domain which affects the channels through activation of G proteins, and these receptors G protein coupled receptors (Purves et al. 2001).
G protein coupled receptors (GPCRs) are the largest family of membrane protein signaling molecules. The activation of these proteins can be achieved by various ligands, thus modulating the activity of a diverse set of signaling pathways (Kobilka 2007). Nowadays, it is estimated that the human organism contains approximately 800 unique GPCRs, and the 460 of them are assumed to be olfactory receptors (Fredriksson et al. 2003). Their main characteristic is that they contain seven transmembrane-spanning segments, which coordinate the position of the protein's N-terminus at the extracellular space and C-terminus at the intracellular space.
Studies in neuromuscular synapse, have demonstrated in detail the neurotransmission mechanism. The binding of the acetylcholine to the postsynaptic receptors, leads to the opening of ion channels. Specifically, the binding of two Ach to a receptor, causes an influx of sodium for milliseconds. In real situations, when an action potential reaches a presynaptic neuron, millions of ACh molecules are secreted into the synaptic cleft. As a result, a high number of AChs will bind to numerous receptors, located on the postsynaptic membrane. The opening of these channels will cause the membrane to depolarize, and the opening of voltage-gated sodium and potassium channels (Purves et al. 2001).
When the postsynaptic membrane potential becomes more negative even from the resting potential, the end plate current increases, and decreases when the membrane potential is more positive. At very positive potential, there is a reverse polarity which cause the current to convert form an inward to an outward one (Purves et al. 2001).
The other category that we will discuss, the hormones, are produced most of the times by specific cells, and initiate a reaction in certain cell types. Only the cells that have hormone receptors, can be used as hormone target cells. These receptors, recognize certain hormones according to their chemical structure. The classes of these receptors are two, the membrane bound receptors, which are transmembrane proteins and the second category the intracellularly localized receptors which are intracellular receptors (Boon 2009).
The membrane bound receptors have an extracellular domain which is linked with an intracellular one. The binding of a hormone are following the rules of noncovalent interactions. In general, signaling molecules for example adrenaline, binds to their receptors with a very high affinity, even higher than the one observed between an enzyme and a substrate. After the binding and the recognition of adrenaline by the receptor, this signal is converted into an intracellular signal, which targets the nuclear compartment. According to the type of the receptors which will bind, adrenaline can cause inhibition of insulin secretion, glycogenolysis and glycolysis (Boon 2009).
Furthermore, the hormone insulin, uses for signal transduction a set of tyrosine kinases receptors. The receptors that have tyrosine kinase (TK) activity (RTKs), contain a specific domain for binding ligand molecules located on the extracellular side. Inside the lipid bilayer there is a single alpha-helical element, and inside the cytosol another part that harbors a TK domain. When the RTKs are not bound with the ligand molecule, most of them are in their monomeric state. The only exception of this paradigm is the insulin receptor and in general its family members. When extracellular stimulus is absent, in our case insulin, the kinase domain of the receptors remains in its inactive, auto inhibited state. In this phase, the receptors either have very low kinase activity, or they haven't at all. After the binding of insulin on the receptor, the consequent transformational changes that lead to the activation of the TK domain. The activation process of TK includes a trans-autosphorylation of tyrosine residues at segments inside and outside of the TK domain. Then, the phosphorylated P-Tyr which is formed at the site of receptor, becomes a docking region for proteins that harbor phosphorylated tyrosine specific domains. Such proteins are SH2, phosphotyrosine-binding (PTB) and C2 .This signaling eventually leads to an increase of the number of glucose transporter 4 in the outer membrane of specific cells, and thus the increased reuptake of glucose from the blood (Boon 2009).
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