A Brief Review on Cell Signaling | Intercellular and Intracellular Signaling

Abstract: Cell signaling is a basic fundamental process through which cells communicate and transcends the information to each other throughout the body. There are several different mechanisms through which every information is transferred in our body and by which many biological functions are carried out. In this review we’ll discuss cell signaling, its types, major cell receptors namely G-protein coupled receptors, ligand gated ion channels, enzyme linked receptors and nuclear receptors. Some signals might be up to a short distance while some might travel a long space by which they are categorized as endocrine and paracrine signaling which is also highlighted in this review.

Keywords: Intercellular signaling, Intracellular signaling, G-protein coupled receptor, Enzyme linked receptor, Ligand gated ion channels, Nuclear receptor

Introduction

Cell signaling 

It can be defined as the ability of a cell to receive and process and transmit the signal. Cellular signaling starts as the first messenger bind to its receptor which can be either embedded on the cell membrane’s outer surface. Due to binding, the cell receptor goes under conformational changes and activates a set of reactions which are then carried out by a secondary messenger.[1]

The cell signaling are basically of two types namely: 

Intracellular and Intercellular

Intercellular cell signaling is a kind of communication in which information is sent from one cell to another. The signals here are sent to either short distance or long distance, and as a result are classified in Autocrine, endocrine, paracrine and synaptic signaling.[2]

Autocrine signaling

The signals have receptors on the signaling cell itself. The signals transmitted by a signaling cell have receptors on their own cell. So the chemicals which are released from the signaling cell, act as a ligand on themselves. Extracellular mediators production and secretion followed by binding of that mediator to the same cell to initiate signal transduction. Example of autocrine signaling can be given as secretion of Interleukin-1 by macrophage and binding of the interleukin-1 by receptor on the macrophage and it results in further activation of these cells and trigger the secretion of more cytokines.[3]

Paracrine signaling

In paracrine signaling, the secretion of mediators from the signaling cell acts on the other cell which is located in its close proximity(15-20 nm distance).[4] This is a very common type of signaling. The signals have a short half-life so they need to act quickly. It eases the organization of localized tissue response such as inflammation and angiogenesis which focuses the action of the mediators within a small region. The signals are diffused passively in the extracellular fluid.[3]

Endocrine signaling 

Signals secreted by the cells are released in the blood circulation and produces effects on the target cells which are located over a longer distance from the signaling cells. The mediators have a comparatively longer half-life than paracrine mediators.

Endocrine and paracrine signaling reflect two extreme paradigms and many mediators show the features that are in between endocrine and paracrine. Cytokines such as IL-1 and TNFα are good example in that they mediate both paracrine and endocrine.[3]

Synaptic signaling

Synaptic signaling occurs when two nerve cells transmit the signal generally from presynaptic neuron to the postsynaptic neuron through a small gap called synapse which is present between signaling cell and receiving cell. Synaptic signaling may also result in the release of paracrine or endocrine mediators by neuroendocrine cells.[3]

Intracellular signaling

Most of the neuronal functions like gene expression, neurotransmission, use-dependent modulation are intracellular signaling initiated at the cell surface.

The molecules associated  with signal transmission and transduction are  profoundly addressed in mammalians  and invertebrates. 

They comprise 4 types of receptor by which signal transduction takes place.

1.  G protein coupled receptor

2.  Ligand gated Ion channel

3.  Enzyme-linked receptor

4.  Nuclear receptor

G-Protein Coupled Receptors

G-protein-coupled receptors, the biggest family of cell-surface molecules involved in signal transmission, are encoded by around 2% of all genes in the human genome.[18] GPCRs are also known as metabotropic receptors or seven transmembrane-domain receptors.[16]

They contain three membrane-bound components

a )  Cell receptor determines to which ligand the cell can respond.

b)   G protein on the intracellular side of the membrane that is stimulated by an activated receptor.

c)   Either end effector enzyme that changes ionic fluxes in the cell in response to activated G protein.[5]

G proteins have the ability to detect the presence of activated receptors and they amplifies the signal by  altering the activity of appropriate effector enzymes and channels. G protein are GTP binding proteins that couple activator of G protein coupled receptor by neurotransmitter at the cell surface to change activity of effector  enzyme. An important protein messenger called cAMP(cyclic Adenosine Monophosphate) synthesized with the help of an effector enzyme called adenylyl cyclase. Second effector enzyme is phospholipase C,  which generates DAG(Diacylglycerol) and IP3(Inositol 1,4,5-triphosphate) which later releases C++ that are stored in the cell.[5][17]

Conversion and activation of G-protein subunits

There are three subunits of G protein namely α, β, and γ.

At the initial stage they are inactive when they are bounded with GDP. They get activated when they are bound with GTP. So to activate the G-proteins, the GDP is to be exchanged with GTP.  When an agonist binds and activates receptor, it goes under conformational changes which causes GDP to dissociate and to be replaced with GTP, this in turn causes dissociation αβγ trimer, which results in formation of α-GTP complex and β-γ complex.  This conversation is one of the key roles of GPCRs.

This conversation is temporary and gets reversed as G proteins have the GTPase enzyme which hydrolyzes GTP and converts G-protein back to GDP bound inactive state.[5]

G proteins are mainly classified into 4 classes: Gs, Gi, Gq, G0. All these G proteins act through different pathways.

Gs

Gs is a stimulatory type of G protein. When a ligand binds to the receptor, it activates the receptor by replacing GDP with GTP. This will allow the formation of the Gα-GTP complex and β-γ complex.  The Gα-GTP complex will move towards the protein called adenylyl cyclase which is located on the cell membrane. The adenylyl cyclase will facilitate the conversion of ATP to Cyclic AMP, an important secondary messenger. This formation of cyclic AMP will allow the activation of Protein Kinase A (PKA). As the word ‘kinase’ indicates it will result in the phosphorylation of proteins. 

Many elements of cellular activity are regulated by cAMP, including enzymes involved in energy metabolism, cell division and differentiation, ion transport, ion channels, and smooth muscle contractile proteins. 

In the liver, fat, and muscle cells, increased cAMP generation in response to β-adrenoceptor activation impacts enzymes involved in glycogen and fat metabolism. As a result, a coordinated response occurs in which glycogen and fat-stored energy is made accessible as glucose to power muscle contraction.

Increased activity of voltage-gated calcium channels in heart muscle cells is a result of cAMP-dependent protein kinases. The amount of Ca2+ entering the cell during the action potential is increased by phosphorylation of these channels, which enhances the heart's contraction force. cAMP-dependent protein kinase phosphorylates (thus inactivates) another enzyme in smooth muscle, myosin light-chain kinase, which is necessary for contraction. This explains why many medicines that stimulate cAMP synthesis in smooth muscle cause smooth muscle relaxation.

The example of this G protein is the mechanism of action of norepinephrine.

Figure 1: Signaling mechanism of different G-proteins

Gi

The mechanism of this G protein is the exact opposite of that of Gs. It's an inhibitory type of G protein. Upon its activation, it inhibits the adenylyl cyclase which results in the inhibition of the conversion of ATP to cyclic AMP. This leads to reduction in protein phosphorylation and decreases heart's contraction. For example, the α-2 stimulation works via. Gi pathway, so it results in the decrease in sympathetic activities and thus lowers the blood pressure. The α-2 receptors work by this mechanism thus inhibiting the release of norepinephrine.

Gq

The Gq mechanism starts with the ligand binding to the GPCR leading to the release of Gα-GTP complex and β-γ complex.

The Gα-GTP complex will further activate the Phospholipase C, which cleaves the phosphatidylinositol bisphosphate which are present in the surface membrane. This in turn generates the secondary messengers called inositol triphosphate (IP3) and Diacylglycerol (DAG). 

IP3 

IP3 is a negatively charged water-soluble molecule that can rapidly diffuse into cytosol to bind with IP3/Ca+2 ligand gated receptors present on the surface of the endoplasmic reticulum. By doing so, it will trigger the release of Ca+2 ions in the cytosol. This calcium will further activate various calcium regulated responses.

DAG

Diacylglycerol is a hydrophobic molecule which diffuses across the cell membrane, interacts and activates a protein called Phospholipase C. This will result in the phosphorylation of proteins and will lead to several cellular responses.

The neurotransmitter glutamate works through this G protein mechanism. 

G0

The G0 is widely expressed in the brain. It also shows the inhibitory action like Gi but through a different mechanism. Upon activation, it shows the inhibitory action of that of Gq by reducing the DAG in cells. They do not show any effect on adenylyl cyclase but they mediate various ion channels such as Ca+2, K+, Na+.

Ligand gated ion channels

Ligand-gated ion channels are a family of transmembrane ion-channel proteins which facilitates the ions such as Na+, K+, Ca2+, and/or Cl to pass through the membrane in response to the binding of a signaling molecule (i.e. a ligand) such as a neurotransmitter.[7] They are the most fastest acting receptor.[6]

When a presynaptic neuron is stimulated, it releases a neurotransmitter into the synaptic cleft from vesicles. After then, the neurotransmitter attaches to receptors on the postsynaptic neuron. If these receptors are ligand-gated ion channels, the ion channels open as a result of the conformational shift, allowing ions to pass across the cell membrane. This causes either a depolarization or a hyperpolarization, depending on whether the receptor is excitatory or inhibitory.

                                                   Figure 2: Illustration of working of ligand-gated ion channels 

Example of these ion channels is a nicotinic acetylcholine(nAch) receptor. The nAch receptor is a pentomer which consists of 4 types of subunit chains namely α, β, γ, and δ. Here there are two α subunits, one β, one γ, and one δ subunit chain. These subunits are nothing but polypeptide chains. The receptor contains the hydrophobic region which is within the membrane and the hydrophilic region which protrudes out on the cytosol part of the cell. 

When the receptor is in a closed state, the hydrophobic region is closely packed, restricting the passage of any ions through the membrane. 

When acetylcholine arrives, it can bind to α-γ binding site or α-δ binding site. When it binds to either site, it creates conformational changes in the structure which results in exposing hydrophilic amino acids and hides the large hydrophobic amino acids thereby opening the receptor and which allows the entry of the ions inside the cell.[8]

Enzyme linked receptor

Extracellular ligands bind to the extracellular side of enzyme-linked receptors, which are also transmembrane proteins. They are a large family, with receptor tyrosine kinases as a main subgroup that phosphorylate the tyrosine residue on the cytosolic side of these proteins. Binding with a triggering signal causes two receptors to bond together, forming a dimer. The dimer's tail sections engage with one another, and each phosphorylates the other.

Protein kinase B, phosphatidylinositol 3-kinase, and other intracellular signaling proteins are attracted to active receptors. The receptor tyrosine kinases, on the other hand, work in a different way. Ras, a tiny protein linked to the cytoplasmic side of the plasma membrane by a lipid tail, is activated by receptor tyrosine kinases. Ras proteins are monomeric GTP-binding proteins with a single unit, analogous to the subunits of trimeric GTP-binding proteins. Ras that has been activated phosphorylates kinase enzymes, which then phosphorylate other kinases, allowing the signal to move from the plasma membrane to the nucleus area. Because mitogen-activated protein kinase (MAP kinase) is the final kinase in the sequence, the entire relay is known as a MAP kinase cascade. The phosphorylation of serine and threonine on transcription-controlling proteins is caused by MAP kinase. The phosphorylation of a gene can either stimulate or decrease its activity. Proliferation, differentiation, and other effects can be seen as a result.

Ras is a crucial protein whose function has been linked to cancer. The cells respond to the stimulation of Ras by growing and proliferating. If Ras continues to be activated, uncontrolled growth and proliferation will occur. A mutation in the Ras gene or other proteins in the system can cause an uncontrollable explosion of growth, which can lead to cancer. 

Information is dissipated through signaling cascades in one way; hormones and local mediators such as cytokines use a different signaling pathway. Their receptors have no inherent enzymatic activity, but they are linked to JAKs, which are cytoplasmic tyrosine kinases.

JAK STATs Signaling Pathway

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is important for signal transmission from cell membrane receptors to the nucleus. The JAK-STAT pathway is required for the production of a wide range of cytokines and growth factors, as well as key cellular activities such hematopoiesis, lactation, and immune system and mammary gland development. Cytokines are important participants in the myeloid and lymphoid lines of cells, and their receptors use this pathway. Type I and type II cytokine receptors bind to a subset of cytokines that includes over 60 different components. These cytokines are required for the induction and coordination of innate and adaptive immunity. Tyrosine kinases that are attached to the cytoplasmic portions of type I and II cytokine receptors are known as JAKs. They were discovered in 1988. The JAK-STAT pathway was coined after three distinct labs found JAKs in 1992. The JAK gets its name from a two-faced Roman god, implying that it has two domains: a catalytic domain and a kinase-like domain. JAKs are inextricably linked to type I and II receptors. When a ligand (cytokine) binds to its receptor, the receptor dimerizes, and JAKs are activated as a result of the close proximity. These activated JAKs induce trans-phosphorylation (also known as transactivation) on certain tyrosine residues, resulting in docking sites for latent cytoplasmic transcription factors known as STATs.[10]

When ligands connect to their receptors, the receptors multimerize. Some receptor subunits are expressed as homodimers, such as erythropoietin and growth hormone, whereas others, such as interferons (IFN) and Interleukins (IL), are expressed as heteromultimers. To induce JAK transphosphorylation and subsequent recruitment of one or more STATs to be phosphorylated, the receptors associated with JAKs must be activated. STATs are proteins that bind to interferon (IFN)-stimulated response regions of DNA sequences to stimulate the transcription of type I IFNs. Phosphorylation is the most prevalent alteration in cell biology, and it is important for the regulation of many signaling pathways. STATs that have not been phosphorylated are found in the cytoplasm. When JAKs are activated, phosphorylation of STATs and STAT dimerization occur, phosphorylated STATs vacate receptor docking sites. As a result, they go to the nucleus and attach to certain DNA regions, either activating or suppressing gene transcription. Although the effect of tyrosine phosphorylation of STATs has been widely documented, the effect of serine phosphorylation of STATs has received less attention. STATs' serine phosphorylation is thought to be independent of their tyrosine phosphorylation.[10]

These are found on the plasma membrane, and when a cytokine binds to its receptor, JAKs are activated, phosphorylate gene regulatory proteins STATs (Signal Transducer and Activator of Transcription), and the process continues. STATs that have been activated move to the nucleus and regulate gene expression by binding to responsive regions, resulting in altered gene expression. Serine/threonine kinases, which are comparable to receptor tyrosine kinases and phosphorylate proteins known as SMADs (Sma and Mad related family proteins), are another method of direct communication. The abbreviation refers to the homologies to the Caenorhabditis elegans SMA ("small" worm phenotype) and MAD family ("Mothers Against Decapentaplegic") of genes in Drosophila.[11]  SMADs are proteins that regulate gene expression, and the TGF-β superfamily frequently uses this pathway.[9]

The JAK-STAT pathway plays an important role in immune system regulation, particularly the fate of T helper cells. T helper cells are crucial in the immunological responses. JAKs are linked to cytokine receptors, which become activated when they are stimulated and phosphorylate STAT proteins, allowing them to be delivered to the nucleus. Because any disruption of the JAK-STAT system and its regulators might result in pathological effects, signaling pathways are prospective therapeutic targets for developing new strategies in the treatment of various diseases, especially disorders which are T cell-mediated.[10]

Nuclear receptor

Steroid hormone receptors are known to act as transcriptional regulators in the nucleus and mediate a variety of important biological processes. Nuclear receptors, particularly steroid hormone receptors, have been studied as models to better understand the principles of transcriptional regulation in mammalian development and adult physiology, as well as how it is affected in disease conditions.[13]

The basic structure of all nuclear receptors is key to understanding their nuclear actions. At the amino terminus, the A and B domains serve as a binding site for transcriptional co-regulators. In the protein interior, the C and D (hinge) regions facilitate DNA binding at defined sequences, are involved in protein nuclear localization, and/or impact tethered binding of the receptor to other transcription factors. Additional co-regulators bind to the E and F domains. A ligand-binding region in the E domain differs structurally in various receptors to confer specificity to ligands and antagonists.[14][15]

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