Cell Signaling Lecture Notes PDF

Cell signaling Lecturer: Dr. Michelle Kuzma Adapted from: Dept. Head, Dr. Danuta Mielżyńska-Švach Molecular biology 2024/2025  Homeostasis Organisms cope with the variability of their external environment by maintaining a relatively stable internal environment. The process by w...

Cell signaling Lecturer: Dr. Michelle Kuzma Adapted from: Dept. Head, Dr. Danuta Mielżyńska-Švach Molecular biology 2024/2025  Homeostasis Organisms cope with the variability of their external environment by maintaining a relatively stable internal environment. The process by which this is done is called homeostasis (homeo - similar, stasis - state). When homeostasis is disrupted, the organism attempts to compensate. If compensation is successful, homeostasis is restored. If compensation is unsuccessful, homeostasis is disrupted, which can result in disease. Homeostasis  Homeostasis Homeostasis is associated with concepts, such as: ❑ extracellular fluid (ECF), which serves as a link between the external environment and cells, ❑ intracellular fluid (ICF), which is located inside cells.  Homeostasis In homeostasis, the composition of both compartments are relatively stable. Since substances are constantly moving between the two compartments, there is a dynamic steady state. A dynamic steady state does not equate to equilibrium because the concentrations of many substances in the ECF and ICF differ; there is rather an established state of imbalance. Established state of imbalance  The role of homeostasis In the case of multicellular organisms, coordination of activities must be ensured on the following levels: ❑ cells within tissues, ❑ tissues within organs and systems, ❑ systems within the entire organism. To maintain homeostasis, cells must cooperate with each other. The cooperation of individual cells and tissues requires the exchange of information carried out through intercellular communication (i.e., cell signaling).  Intercellular communication Intercellular communication is essential for: ❑ cell survival, ❑ cell division, ❑ cell differentiation, ❑ cell death.  Intercellular signaling Coordination between cells involves the transmission of signals. There are two basic types of signals: ❑ electrical - related to changes of the cell's membrane potential ❑ chemical - chemical compounds (i.e., molecules) secreted by cells into the extracellular space Cells that respond to electrical or chemical signals are called the target cells.  Methods of communication There are two types of communication depending on the distance the signal travels to reach a target cell: ❑ local ❑ distant Types of local communication: ❑ juxtacrine (contact-dependent) ❑ paracrine ❑ autocrine Types of distant communication: ❑ endocrine (hormonal) ❑ neuronal communication  Types of cell communication Juxtacrine/contact-dependent cell-cell communication requires direct contact between cells Direct transfer of molecules occurs via gap junctions between adjacent cells.  Types of cell communication Juxtacrine communication (direct contact) A signaling molecule on the surface of the cell membrane of one cell binds to a receptor on the cell membrane surface of another cell. Cellular receptors: specialized proteins capable of receiving, transforming and transmitting information from the external environment to effectors in the cell.  Types of cell communication Paracrine communication Molecules released by a cell into the extracellular fluid act on neighboring cells  Types of cell communication Autocrine communication Molecules released into the intercellular fluid act on the same cell that secreted them.  Types of cell communication Endocrine communication The endocrine system communicates through hormones (hormone - to stimulate), which are chemical compounds secreted into the blood and distributed throughout the body by the circulatory system. Hormones contact most of the body's cells, but only some are target cells. Types of cell communication  Types of cell communication Neuronal (synaptic) communication The nervous system uses a combination of electrical and chemical signals to communicate over long distances. The electrical signal travels along the neuron to its end where it is converted into a chemical signal. Neurons communicate with each other via synapses. Neuronal communication  Neuronal communication Chemicals secreted by neurons are called neurocrine molecules. Types of neurocrine molecules are: ❑ neurotransmitters that only diffuse across the synaptic cleft and therefore, have a fast effect, ❑ neurohormones that diffuse into the bloodstream and are distributed throughout the body thereby, having a relatively slower effect. Neuronal communication Examples of signaling molecules Also a neurotransmitter  Cells communication principles Only cells that have receptors for signaling molecules respond to a given signal (selective response). The response of a cell to a signal depends on the functional specialization of the cell and on receptor type (NOT the signaling molecule itself): ❑ there may be different receptors for one signaling molecule, ❑ one signaling molecule may induce different changes in different target cells, respectively, ❑ one signaling molecule may be able to induce many different changes in a given target cell. Cells communication principles Acetylcholine can cause a variety of reactions.  Intercellular communication Signaling molecules that bind to receptors are known as ligands and are categorized by how they interact with a given receptor: ❑ agonists: stimulate the receptor ❑ antagonists: inhibit the receptor  Intercellular communication Ligands are also called extracellular signaling molecules because they deliver information to a target cell. Binding of the ligand to the receptor upregulates, downregulates, partially activates the receptor. The receptor, in turn, activates one or more intercellular signaling molecules. The final signaling molecule triggers the final response, which may be protein modification or synthesis.  Types of signaling molecules Signaling molecules are divided into: ❑ those that pass through the cell membrane because they are small enough and/or hydrophobic; they require the presence of an intracellular receptor in the target cell, ❑ those that do not pass through the cell membrane because they are large and/or too hydrophilic; they require the presence of cell-surface receptors on the surface of the target cell.  Types of receptors Intracellular receptors are divided into: ❑ cytoplasmic receptors (Type I NR) ❑ nuclear receptors (Type II NR) Cell-surface receptors are divided into: ❑ ion channel-linked receptors, ❑ G protein-coupled receptors, ❑ enzyme-linked receptors. Types of receptors  Intracellular receptors Intracellular receptors are transcription factors located in the cytoplasm as well as in the nucleus. Respective ligands are small and hydrophobic molecules that can diffuse through the cell membrane, such as: ❑ steroid hormones, ❑ thyroid hormones, ❑ vitamin D, ❑ retinoic acid.  Intracellular receptors In the active state (ligand bound to receptor), intracellular receptors are only in the nucleus and participate in the regulation of gene expression (i.e., transcription)  Intracellular receptors Nuclear receptors are composed of several elements, such as: ❑ a ligand binding domain (LBD), ❑ a DNA binding domain (DBD), ❑ a hinge region that controls the movement of the activated receptor into the nucleus, ❑ a transcription-activating domain.  Intracellular receptors Intracellular receptors bind to the HRE (hormone response element) sequence in target genes. The HRE sequence consists of 15 nucleotides. These sequences are usually located within the promoter, but sometimes they are located at a distance from it.  Intracellular receptors Some nuclear receptors are monomeric. Most nuclear receptors are dimeric. The dimers can be either homodimers or heterodimers. Monomer Homodimer Hetreodimer  Intracellular receptors Intracellular receptor families include receptors: ❑ for lipophilic hormones, ❑ for active vitamin A (retinol), ❑ for active vitamin D3 (1,25-dihydroxycholecalciferol), ❑ for ligands that have yet to be identified ("orphan" receptors)  Steroid hormones The steroid hormone cortisol (involved in stress response) activates a target gene transcription regulator.  Cell-surface receptors Cell-surface receptors are proteins located on/within the membrane of the target cell. The ligands are large, hydrophilic and/or charged molecules that cannot diffuse through the cell membrane. All cell-surface receptor proteins bind to an extracellular signal molecule and transduce its message into one or more intracellular signaling molecules that alter the cell’s behavior. Cell-surface receptors Types of cell-surface receptors  Ion channel-linked receptors The binding of a ligand to a receptor causes a change in the conformation of the proteins that make up the ion channel. The membrane potential changes, which affects the permeability of the cell membrane to specific ions. Ions pass through the open channel according to the concentration gradient (Na+, K+, Ca2+, Cl-). Most receptors for neurotransmitters have this characteristic (fast response).  Mechanism of action Binding of a ligand to a cell-surface receptor Change in receptor conformation Depolarization or hyperpolarization of the cell membrane Creation of an action potential Channel opens and ion flow occurs with the concentration gradient Na+ K+ Na+ K+  Ion channel-coupled receptors The type of ion channel depends on the type of stimuli: ❑ voltage-gated ❑ ligand-gated by an extracellular ligand ❑ ligand-gated by an intracellular ligand ❑ mechanically gated An important feature of ion channels is selectivity (i.e., the ability to pass strictly defined types of ions through): ❑ cationic or anionic ❑ "specialized" - sodium, potassium, calcium, etc. Ion channel-coupled receptors  Ion channel-coupled receptors A voltage-gated ion channel changes conformation depending on the membrane potential: ❑ the channel is closed when the cell is at rest and the cell membrane is polarized, ❑ the channel is open when the cell membrane is depolarized, ❑ after a period of opening, the channel is temporarily inactivated and cannot open (refractory period), ❑ after repolarization of the cell membrane, the channel returns to its initial conformation, closed. Ion channel-coupled receptors  Ion channel-coupled receptors Ligand-gated ion channels activated by neurotransmitters convert chemical signals in the target cell into electrical signals. The ion channels open after binding a neurotransmitter, which causes a change in the permeability of the cell membrane to ions. As a result, the following occurs: ❑ a change in membrane potential, ❑ depolarization of the cell membrane, ❑ generation of an action potential. Ion channel-coupled receptors Ion channel-coupled receptors  G protein-coupled receptors G protein-coupled receptors (GPCRs) are receptors that combine with ligands to transmit a signal to secondary signaling molecules – signal transduction. Such signal transmission is usually much slower and more complex than via ion-channel-coupled receptors. GPCR effects last longer. GPCRs constitute the largest group of receptors.  Signal transduction Signal transduction consists of: ❑ the signaling cell sending a so-called ligand (i.e. an extracellular signaling molecule like a hormone or neurotransmitter), ❑ the ligand binds to a specific receptor present in the target cell membrane, ❑ intracellular signaling molecules are produced inside the cell. This initiates a series or cascade of reactions in which the final signal is transmitted to the effector protein. Signal transduction Cascade and amplification Cascade Amplification  Molecular relay race The signaling process inside a cell is often referred to as "a molecular relay race". Information is passed from one signaling molecule to another. These successive steps continues until: ❑ an enzyme involved in metabolism is activated or inactivated, ❑ the cytoskeleton acquires a new configuration, ❑ a specific gene is transcribed or turned off.  Molecular switches Many signaling molecules act as molecular switches because receiving a signal switches them from an inactive to an active state. When activated, they can turn on or inhibit other proteins in the signaling pathway. These proteins remain active until another process turns them off.  Molecular switches Molecular switches are divided into two classes: ❑ proteins whose activity are turned on or turned off by adding or removing phosphate groups (i.e., phosphorylation or dephosphorylation, respectively) ❑ proteins whose activity depends on the exchange of bound GDP (guanosine-5′-diphosphate) for GTP (guanosine-5′- triphosphate) and vice versa Types of molecular switches G protein-coupled receptors  7TM proteins GPCRs are the largest family of cell-surface receptors (there are over 700 in humans). Despite the diversity of signaling molecules that bind to them, all GPCRs have the structure of 7TM receptors. 7TM receptors consist of seven subunits (α-helices) that span the lipid bilayer of the cell membrane. 7TM receptors have: ❑ an extracellular ligand-binding domain, ❑ an intracellular G protein-binding domain. When a ligand binds to a 7TM receptor, a conformational change is induced.  7TM protein N C  G proteins G proteins are a group of proteins bound to guanosine-5′- diphosphate, known as GDP. G proteins are located on the cytoplasmic side of the cell membrane. G proteins are composed of three protein subunits (domains): ❑ alpha (α) ❑ beta (β) ❑ gamma (γ)  Protein kinase Protein kinases are a group of enzymes that phosphorylate proteins specific to a given kinase. Phosphorylation leads to a change in the conformation of the target protein, and therefore, can: ❑ change its activity, ❑ change its ability to bind to other proteins, ❑ cause the molecule to move within the cell. ~30% of proteins are regulated in this way. Most metabolic pathways of the cell, especially in cell signaling, involve enzymes from the protein kinase group.  G proteins There are different types of G proteins that are specific for different receptors and target proteins. They act as intermediaries and carry the signal from the modified 7TM receptor to the effector. The type of α-subunit present is most important for the specificity of signal transduction. G proteins based on the α subunit are divided into: ❑ stimulating (Gs) ❑ inhibitory (Gi)  Mechanism of action In the resting state, 7TM receptors and G proteins are inactive. Binding of a ligand to the membrane receptor causes a change in the conformation of the: ❑ 7TM receptor on the extracellular side of the membrane, ❑ G protein on the cytoplasmic side of the membrane. In the inactive G protein, GDP is linked to the alpha (α) subunit.  Mechanism of action Activation of the G protein involves the exchange of GDP for GTP in the α-subunit. This breaks up the G protein into two molecules: ❑ the α-subunit linked to GTP, ❑ The G βγ complex. The active α-subunit and the G βγ complex can interact with a specific target protein in the plasma membrane. Mechanism of action Mechanism of action α-subunit Mechanism of action βγ complex  Second messenger molecules The targets of G proteins are most often proteins that are located within the cell membrane. Examples include: ❑ adenylyl cyclase ❑ phospholipase These proteins catalyze the formation of second messenger molecules that are located inside of the cell. Adenylyl cyclase is responsible for the formation of cyclic adenosine-3′,5′-monophosphate (cAMP). Phospholipase C is responsible for the formation of molecules: ❑ inositol trisphosphate (IP3), ❑ diacylglycerol (DAG).  Adenylyl cyclase Ligands that cause activation of adenylyl cyclase are adrenaline, acetylcholine, and glucagon. Stages of adenylyl cyclase action are: ❑ the ligand binds to the 7TM receptor, ❑ the G protein is activated, ❑ the α-subunit of the G protein binds to adenylyl cyclase, ❑ the active adenylyl cyclase synthesizes cyclic adenosine- 3′,5′-monophosphate (cAMP). Adenylyl cyclase Adenylyl cyclase  Cyclic AMP Stages of cyclic AMP activity: ❑ activation of protein kinase A (PKA) ❑ activation of other proteins responsible for: ❑ glycogen breakdown (e.g., in muscle cells), ❑ regulation of gene expression (e.g., in endocrine cells of the hypothalamus), ❑ others. Protein kinase A Protein kinase A  Phospholipase C Ligands that cause activation of phospholipase C are acetylcholine, vasopressin and thrombin. Stages of phospholipase C activity: ❑ a ligand binds to the 7TM receptor ❑ the G protein is activated ❑ the α-subunit binds to phospholipase C ❑ activation of phospholipase C and breakdown of phosphatidylinositol (inner part of the cell membrane) ❑ formation of second messenger molecules: ❑ inositol triphosphate (IP3) -> diffuses into cytosol ❑ diacylglycerol (DAG) -> remains membrane-bound  Phospholipase C Action of inositol triphosphate (IP3): ❑ binds to the Ca2+ channel in the ER membrane and opens it ❑ an electrochemical gradient causes an efflux of Ca2+ ions from the ER to the cytosol Action of diacylglycerol (DAG): ❑ together with Ca2+ ions, participates in the activation of protein kinase C (PKC) in the cytosol ❑ PKC moves from the cytosol to the cytoplasmic side of the cell membrane ❑ active PKC participates in the phosphorylation of other proteins inside the cell. Phospholipase C  Enzyme-linked receptors Enzyme-linked receptors are receptors that act as enzymes or bind to proteins that become enzymes. Enzyme-linked receptors usually have only one transmembrane segment, which spans the lipid bilayer as a single α-helix that: ❑ binds to a ligand on the outside of the cell membrane, ❑ has a catalytic center or an enzyme- binding site on the inside of the cell membrane. Enzyme-linked receptors  Enzyme-limkedreceptors Enzyme-linked receptors: ❑ are active at low ligand concentrations, ❑ have slow subsequent responses (on the order of hours), ❑ have effects that may require many intracellular transduction steps that usually lead to a change in gene expression.  Enzyme-linked receptors Enzyme-linked receptors are divided into receptors with the following functions: ❑ tyrosine kinase ❑ serine-threonine kinase ❑ guanylate cyclase  Tyrosine kinase Growth factors (i.e., FGF, EGF, PDGF, VEGF) are the ligands that cause activation of tyrosine kinases. Stages of tyrosine kinase action: ❑ a ligand binds to a receptor (active site), ❑ connection of the membrane of two receptor molecules to form a dimer, ❑ stimulation of kinase activity - a mutual phosphorylation of tyrosine residues, ❑ binding of signaling proteins, ❑ phosphorylation of subsequent signaling proteins - transmission and/or amplification of a signal in the cell. Activation of tyrosine kinase  Ras protein Most tyrosine kinases activate Ras proteins. The Ras protein is: ❑ a monomeric G protein (GTPase), ❑ bound to the cell membrane by a membrane lipid, ❑ occurs in the following states: ❑ inactive (bound to GDP) ❑ active (bound to GTP) Ras protein activation  Ras protein activation An adaptor protein is attached to a specific phosphorylated tyrosine residue. The adaptor protein then binds to the GEF protein, forming a Ras-GEF complex. The Ras-GEF complex forces the Ras protein to convert GDP to GTP (i.e., causing its activation). Tyrosine kinase - Ras protein  The phosphorylation cascade The activated Ras protein triggers a phosphorylation cascade of three serine-threonine kinases that carry a signal further. Individual kinases in the cascade are phosphorylated and activated by enzymes called kinase kinases. The final kinase in the cascade is called the MAP kinase (mitogen-activated protein kinase) and is involved in the phosphorylation of further signaling or target proteins. The Ras protein - MAPK pathway  PI3K/Akt pathway Stages of growth and survival signaling (e.g., IGF): ❑ activation of tyrosine kinase, ❑ binding and activation of PI3K, ❑ PI3K phosphorylates phosphatidylinositol, ❑ binding of phosphatidylinositol to protein kinase B (PKB/Akt), ❑ Akt is phosphorylated by protein kinase 1 and protein kinase 2, ❑ release of active Akt from the cell membrane, ❑ active Akt then participates in the phosphorylation of other proteins inside the cell. PI3K/Akt pathway PI3K/Akt/mTor pathway Signal transduction  Cell signal response Extracellular signals can trigger fast or slow responses. Motility changes, secretion and metabolism require changes in protein function and therefore, occur rapidly. Cell growth, differentiation, or division require the synthesis of new proteins and changes in gene expression and therefore, occur relatively slowly. Signal integration Cell signal response  Literature Essential of Cell Biology B. Alberts, D. Bray, K. Hopkin. Volume 2:Chapter 16. Cell Signaling