Physiology 210 Cell Physiology - C1 - 2024 PDF

Physiology 210 Cell Physiology Dr. Robin Clugston Ph.D. [email protected] https://giphy.com/gifs/13nmmdyzCEGXZu (8/2018) Copyrighted material contained herein is reproduced under the fair dealing exception of the Canadian Copyright Act. This document...

Physiology 210 Cell Physiology Dr. Robin Clugston Ph.D. [email protected] https://giphy.com/gifs/13nmmdyzCEGXZu (8/2018) Copyrighted material contained herein is reproduced under the fair dealing exception of the Canadian Copyright Act. This document was prepared by Robin Clugston. It is intended for the individual use of registered students only, and not for wider distribution. Outline of Lecture series Part 1: Cellular Structure Part 2: Membrane transport Part 3: Cellular signaling Why start with cells? neurophysiology renal endocrinology Basic cell physiology gastrointestinal cardiovascular respiratory The human body is comprised of ~200 distinct types of cells There are four broad categories of cells: 1. Epithelial cells 2. Connective tissue cells 3. Nerve cells 4. Muscle cells We will focus on the physiology of “generic” cells Cells are surrounded by membranes and contain specialized structures called organelles Lecture outline: 1. Organelles 2. Membranes Cells contain specialized structures called organelles Vander, Figure 3.2 Cells contain specialized structures called organelles Nucleus Ribosomes Endoplasmic reticulum Golgi Apparatus Endosomes Vander, Figure 3.3 Mitochondria Lysosomes Peroxisomes Cytoskeleton Cellular organelles: the nucleus Cellular organelles: the nucleus - Largest organelle (typically one/cell) - Primary function: storage of genetic information (DNA!), in the form of chromatin - Surrounded by membrane = nuclear envelope, with openings called nuclear pores - Nuclear pores facilitate… - Passage of RNA into the cytoplasm - Entrance of proteins that modulate gene expression - Contains nucleolus: site of ribosomal RNA synthesis and protein components of ribosomes Vander, Figure 3.10 Cellular organelles: ribosomes - Smallest (~20 nm) and most abundant organelle (~10 million) - Primary function: “protein factory” – translates RNA into protein - No surrounding membrane (comprised of proteins and RNA) - Critical component of the central dogma of molecular biology (Watson & Crick) DNA >>>> RNA >>> PROTEIN nucleus ribosome - Found floating free in the cytoplasm or attached to endoplasmic reticulum - Free ribosomes primarily synthesize cytosolic proteins - Membrane-bound ribosomes primarily synthesize membrane bound proteins Cellular organelles: endoplasmic reticulum - Comprises extensive network of membrane-enclosed space distributed throughout the cell - Two forms: - smooth endoplasmic reticulum - Rough endoplasmic reticulum Smooth ‘ER’ - continuous with rough ER and nuclear envelope - Contains enzymes associated with for e.g. fatty acid synthesis. Stores and releases calcium* - Rough ‘ER’ - “rough” appearance comes from adherent ribosomes - Site of protein synthesis. Proteins synthesized in lumen distributed to other organelles or secreted Vander, Figure 3.11 Cellular organelles: Golgi apparatus - Structure: series of membranous sacs (“cisternae”), forming a cup shape. Polar, with a “cis” and “trans” face. - Function: cellular “post office”; modifies and sorts proteins arriving from the rough ER; distributes them to other organelles or to the membrane for secretion trans face cis face Vander, Figure 3.12 Cellular organelles: Golgi apparatus - Structure: series of membranous sacs (“cisternae”), forming a cup shape. Polar, with a “cis” and “trans” face. - Function: cellular “post office”; modifies and sorts proteins arriving from the rough ER; distributes them to other organelles or to the membrane for secretion Lysosomes Membrane proteins Secretion Rough ER Golgi Cellular organelles: endosomes Structure: small membrane-bound vesicle Function: Sorting vesicular “traffic” in the cell. https://micro.magnet.fsu.edu/cells/endosomes/endosomes.html Endocytic Early Late Lysosome vesicle endosome endosome Cellular organelles: Lysosomes and Peroxisomes Lysosomes Peroxisomes Small, membrane-bound vesicles Cellular “stomach” Cellular “reprocessing plant” Acidic environment, containing digestive Neutral pH, contain oxygen-consuming enzymes enzymes, which generate hydrogen peroxide Breakdown: Breakdown: - damaged/worn out organelles - Fatty acids (beta-oxidation) - Engulfed bacteria - Detoxification of alcohol (& other - Engulfed debris of dead cells toxins) Important in body’s defence systems Cellular organelles: mitochondria Structure: double-membrane bound, interconnected rod-like structures. Inner membrane folded into “cristae”, giving distinct appearance Function: “powerhouse” of the cell; transfers energy present in nutrients to adenosine triphosphate (ATP) in a process called cellular respiration Vander, Figure 3.13 Fun fact: is it thought that mitochondria evolved from prokaryotic cells that started living inside eukaryotic cells (endosymbiotic hypothesis) Cellular organelles: Cytoskeleton Structure: Network of protein filaments throughout cell Function: provides structural support, facilitates changes in cell shape, produces cell movement Three classes: 1. Actin filaments (red) 2. Intermediate filaments 3. microtubules (green) Cellular organelles: Cytoskeleton Structure: Network of protein filaments throughout cell Function: provides structural support, facilitates changes in cell shape, produces cell movement Three classes: 1. Actin filaments 2. Intermediate filaments 3. microtubules G-actin subunits form polymer of two Actin filament twisting chains, forming F-actin Intermediate Twisted strands of multiple possible filament proteins, e.g. keratin, desmin, laminin Hollow tubes, formed from tubulin microtubules subunits Cellular organelles: Cytoskeleton Structure: Network of protein filaments throughout cell Function: provides structural support, facilitates changes in cell shape, produces cell movement Three classes: 1. Actin filaments 2. Intermediate filaments 3. microtubules Actin filament Intermediate filament microtubules Vander, Figure 9.2 Cellular organelles: Cytoskeleton Structure: Network of protein filaments throughout cell Function: provides structural support, facilitates changes in cell shape, produces cell movement Three classes: 1. Actin filaments 2. Intermediate filaments 3. microtubules Actin filament Intermediate filament microtubules Cellular organelles: Cytoskeleton Structure: Network of protein filaments throughout cell Function: provides structural support, facilitates changes in cell shape, produces cell movement Three classes: 1. Actin filaments 2. Intermediate filaments 3. microtubules Actin filament Intermediate filament microtubules Cells are surrounded by membranes and contain specialized structures called organelles Lecture outline: 1. Organelles 2. Membranes Cells and their internal structures are surrounded by membranes Vander, Figure 3.5a The fluid-mosaic model describes plasma membrane structure Vander, Figure 3.8 The plasma membranes contain phospholipids, cholesterol and proteins The phospholipid bilayer Phospholipids are amphipathic hydrophilic head hydrophobic tail Vander, Figure 3.5b The plasma membranes contain phospholipids, cholesterol and proteins Membrane cholesterol Found primarily in the outer cell membrane (very little in intra- cellular membranes) non-polar polar Vander, Figure 2.13b Vander, Figure 3.5b Cholesterol is slightly amphipathic Functions to maintain membrane fluidity The plasma membranes contain phospholipids, cholesterol and proteins Membrane proteins Two classes: 1. Integral membrane proteins 2. Peripheral membrane proteins Vander, Figure 3.5b The plasma membranes contain phospholipids, cholesterol and proteins Membrane proteins Two classes: 1. Integral membrane proteins Embedded in the membrane or membrane spanning (so-called “transmembrane” proteins) Amphipathic (contain polar and non-polar domains/amino acids) Vander, Figure 3.5b Vander, Figure 3.7 The plasma membranes contain phospholipids, cholesterol and proteins Membrane proteins Two classes: 1. Integral membrane proteins Embedded in the membrane or membrane spanning (so-called “transmembrane proteins) Amphipathic (contain polar and non-polar domains/amino acids) 2. Peripheral membrane proteins Not amphipathic Vander, Figure 3.5b Lie on membrane surface, bound to polar regions of integral proteins Primarily on cytosolic surface Structure-Function relationship of cell membranes 1. Link adjacent cells together (Part 1) 2. Regulate passage of substances into and out of cell (Part 2) 3. Detect chemical messengers arriving at the cell surface (Part 3) 4. Anchor cells to extracellular matrix Cell membranes can join together to form junctions - Important factor in the formation of tissues - There are three major types of cell junctions 1. Desmosomes 2. Tight junctions 3. Gap junctions - Defined by unique structural and functional properties - Mediated by distinct transmembrane proteins Vander, Figure 3.9 Cell membranes can join together to form junctions - Important factor in the formation of tissues - There are three major types of cell junctions 1. Desmosomes 2. Tight junctions 3. Gap junctions Structural characteristics: - Adjacent cells separated by ~ 20 nm - Form “dense plaques” - Firm attachment between cells gives structural integrity Protein components: - Cadherins (extend into extracellular space and bind with cadherins from adjacent cells) - Keratin (anchors desmosome to cytoskeleton) Vander, Figure 3.9 Cell membranes can join together to form junctions - Important factor in the formation of tissues - There are three major types of cell junctions 1. Desmosomes 2. Tight junctions 3. Gap junctions Structural characteristics: - No space between adjacent cells - Occurs in band around entire cell - Common in epithelia Protein components: - Complex >40 known proteins - Occludins - Claudins Vander, Figure 3.9 Cell membranes can join together to form junctions - Important factor in the formation of tissues - There are three major types of cell junctions 1. Desmosomes 2. Tight junctions 3. Gap junctions Structural characteristics: - Adjacent cells separated by ~ 2-4 nm - Form pores between cells, allowing passage of ions and small molecules Protein components: - Connexins Vander, Figure 3.9 Learning outcomes At the end of Part 1, you should be able to identify and describe the structural components of cells: 1. Membranes Outline the basic structure of the plasma membrane Define the molecules that make up cell membranes (e.g. lipids and proteins), and explain their contribution to membrane structure and function Describe the three types of membrane junctions, their function, and defining structural properties 2. Organelles Describe the basic structure and function of cellular organelles Nucleus Ribosomes Endoplasmic reticulum Golgi Apparatus Endosomes Mitochondria Lysosomes Peroxisomes Cytoskeleton Physiology 210 Cell Physiology Part 1: Cellular Structure Part 2: Membrane transport Part 3: Cellular signaling Part 2: membrane transport Movement of molecules across the cell membrane is critical to maintain cellular homeostasis Outline: molecules can cross membranes through multiple mechanisms 1. Diffusion Diffusion 2. Protein-mediated transport 3. Vesicular transport 4. Epithelial transport Diffusion through membranes Diffusion across the membrane can occur through the lipid bilayer, or via proteins The polarity of molecules determines their ability to diffuse across the membrane - Nonpolar molecules diffuse across membranes relatively quickly (e.g. oxygen, carbon dioxide, fatty acids) - Polar molecules diffuse across membranes relatively slowly (e.g. ions [K+], glucose) Hydrophobic interior Impedes diffusion (nonpolar) Vander, Figure 3.8 Diffusion through membranes Diffusion across the membrane can occur through the lipid bilayer, or via proteins Diffusion through the lipid bilayer Vander, Figure 3.8 Diffusion through membranes Simple diffusion reflects the random motion of molecules leading to their even distribution Vander, Figure 4.1 Diffusion through membranes Simple diffusion reflects the random motion of molecules leading to their even distribution “diffusion equilibrium” Vander, Figure 4.2 Diffusion through membranes The direction of diffusion is a product of the balance between one-way flux between compartments, and is referred to as net flux Vander, Figure 4.3 Diffusion through membranes Diffusion through membranes can be described by Fick’s first law of diffusion Permeability coefficient, factor Rate of diffusion unique to a given molecule at a (magnitude of net flux) given temperature Different molecules diffuse at J = PA(Co - Ci) different rates Difference in concentration Surface area of the membrane (C) between the outside (o) Greater surface area = and inside (i) of the cell great area for diffusion to take place, therefore faster Large differences in net flux concentration will drive greater diffusion Outline: molecules can cross membranes through multiple mechanisms 1. Diffusion 2. Protein-mediated transport 3. Vesicular transport 4. Epithelial transport Diffusion Protein-mediated transport Vander, Figure 4.10 Facilitated diffusion - Diffusion cannot account for all movement of molecules across membranes - Protein-mediated movement of ions, amino acids and other small molecules also occurs - Note: ion channels & membrane transporters are selective toward specific molecules - Two broad categories: 1. Ion channels (facilitate diffusion of ions across membrane) 2. Transporters (facilitate movement of specific solutes across membranes) Protein-mediated transport - Major difference between ion channels and transporters is in the number of molecules that can cross >>> ion channels typically move 1000X more molecules than transporters Ion channel ferry Diffusion through ion channels - Ions (Na+, K+, Cl- and Ca2+) diffuse across cell membranes through transmembrane proteins called ion channels - Two forces contribute to the flux of ions across membranes - Ion concentration (chemical) - Electrical gradient between outside and inside of cell (membrane potential) - Taken together, these forces are described as the electrochemical gradient across a membrane Vander, Figure 4.6 Diffusion through ion channels - Ion channels are transmembrane proteins that form pores - The small diameter of ion channel pores prevents larger molecules from passing through - Ion channels show selective permeability to specific ions, this selectivity is determined by: 1) channel diameter 2) charge of the polypeptides 3) number of water molecules associated with ion Vander, Figure 4.5 Diffusion through ion channels - Ions can diffuse across cell membranes through ion channels - Ion channels are transmembrane proteins than form pores - Diffusion of ions through ion channels is controlled by a process called channel gating 1. ligand-gated: binding of specific molecule to channel causes conformational change 2. voltage-gated: a change in membrane potential causes conformational change 3. Mechanically-gated: a physical change in the membrane (e.g. stretch) Facilitated diffusion - Diffusion cannot account for all movement of molecules across membranes - Protein-mediated movement of ions, amino acids and other small molecules also occurs - Note: ion channels & membrane transporters are selective toward specific molecules - Two broad categories: 1. Ion channels (facilitate diffusion of ions across membrane) 2. Transporters (facilitate movement of specific solutes across membranes) Membrane transporters - The general model for protein-mediated transport across membranes includes three steps: 1) Solute binding to specific site on protein surface exposed to extracellular fluid 2) Conformation change in transporter, exposing bound solute to intracellular fluid 3) Dissociation of solute from binding site into the intracellular fluid Vander, Figure 4.8 (Note – process can be in reverse too) Membrane transporters - Like ion channels, membrane transporters are selective toward specific molecules - There are many different kinds of transports, e.g. the solute carrier group of transport proteins has over 400 members - Different cells express different transports, depending on their function - Magnitude of flux through transporters dependent on four factors: 1) solute concentration 2) affinity of transporter for solute 3) numbers of transporters in the membrane 4) rate at which the transporter goes through conformational change AA AA AA AA AA AA AA AA AA AA AA AAAA AA AA AA AA AA AA AA AA AA AA AA AA AA AA Membrane transporters - Cells only express a finite number of transporters, which can become saturated, therefore limiting the rate of flux into the cell AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA - Thus, for diffusion, flux into cells is directly proportional to extracellular concentration and is relatively limitless - Mediated transport is limited by transport factors discussed on last slide Vander, Figure 4.9 Membrane transporters - Previous discussion included general model of protein-mediated transport, but actually two physiological types of transport exist: 1) facilitated diffusion 2) active transport “downhill” via membrane protein - ‘stop’ when concentration equalized - does not require ATP “uphill” via membrane protein - often referred to as “pumps” - Requires energy to overcome concentration difference Vander, Figure 4.10 Protein-mediated transport > active transport - Energy required to power active transport can comes from two known sources: 1) the direct use of ATP > primary active transport 2) the use of an electrochemical gradient across a membrane > secondary active transport Protein-mediated transport > primary active transport - Requires ATP - “ATPase” transporters hydrolyse ATP 1) provides energy 2) phosphorylates protein, a covalent modulation that changes protein confirmation - Best-studied example of a primary active transporter is the Na+/K+-ATPase pump - present in all cells - moves Na+ from intracellular to extracellular fluid - moves K+ from extracellular to intracellular fluid - Maintains relatively high levels of intracellular K+, and low Na+ - Moves three Na+ ions out of the cell and brings in two K+ ions = net transfer of positive charge Protein-mediated transport > primary active transport - Na+/K+-ATPase pump - Model for Na+/K+-ATPase pump action involves five steps 1) 3 sodium ions bind to high-affinity binding sites on intracellular surface of ATP-bound protein (Potassium ions will not bind to their binding sites because they are in a low-affinity state Protein-mediated transport > primary active transport - Na+/K+-ATPase pump - Model for Na+/K+-ATPase pump action involves five steps 2) Sodium binding activates ATPase activity of transporter. Surface of protein is phosphorylated and ADP is released Protein-mediated transport > primary active transport - Na+/K+-ATPase pump - Model for Na+/K+-ATPase pump action involves five steps 3) Protein phosphorylation causes conformational change, exposing bound sodium to the extracellular environment. Conformational changes also changes affinity for sodium, releasing it. Protein-mediated transport > primary active transport - Na+/K+-ATPase pump - Model for Na+/K+-ATPase pump action involves five steps 4) Conformational change increases affinity for potassium, which bind to the extracellular surface of the protein, triggering release of phosphate Protein-mediated transport > primary active transport - Na+/K+-ATPase pump - Model for Na+/K+-ATPase pump action involves five steps 5) Release of phosphate returns protein to original confirmation, resulting in reduced affinity for potassium and its release into the cell. Protein-mediated transport > active transport - Energy required to power active transport can comes from two known sources: 1) the direct use of ATP > primary active transport 2) the use of an electrochemical gradient across a membrane > secondary active transport Protein-mediated transport > secondary active transport - Movement of ion down electrochemical gradient coupled to transport of other (organic) molecule, such as glucose or an amino acid - Example: - Sodium flows along concentration gradient into cell (“downhill”) - Solute pulled inside against its concentration gradient (“uphill”) - Cycle of steps similar to primary active transport, except no ATP consumed - (Maintenance of sodium’s concentration gradient depends on primary active transport) Vander, Figure 4.13 Outline: molecules can cross membranes through multiple mechanisms 1. Diffusion 2. Protein-mediated transport 3. Vesicular transport 4. Epithelial transport Vesicular transport is a specialized mechanism to transport molecules across membranes - Does not require molecules to pass through membrane – transport is achieved by enclosure into vesicles that pinch off from the membrane 1) Endocytosis: membrane envaginations enclose small volume of extracellular fluid, which are taken into the cell 2) Exocytosis: intracellular membrane-bound vesicles fuse with the plasma membrane and release their contents into the extracellular fluid Vander, Figure 4.20 Vesicular transport across membranes: endocytosis - There are three common types of endocytosis: 1) Pinocytosis 2) Phagocytosis 3) Receptor-mediated endocytosis Vesicular transport across membranes: endocytosis - There are three common types of endocytosis: 1) Pinocytosis (aka fluid endocytosis) 2) Phagocytosis 3) Receptor-mediated endocytosis - Nonspecific - includes water and whatever solutes are present - Vesicle fuses with lysosome, where contents are hydrolyzed Vesicular transport across membranes: endocytosis - There are three common types of endocytosis: 1) Pinocytosis 2) Phagocytosis 3) Receptor-mediated endocytosis - Specific (involves interaction between particle and cell surface) - Unique to specialized cells of the immune system (phagocytes) - Involves uptake of bacteria or cell debris from damaged tissue - Internalized vesicle called a phagosome, fuses with lysosome, where contents are hydrolyzed Vesicular transport across membranes: endocytosis - There are three common types of endocytosis: 1) Pinocytosis 2) Phagocytosis 3) Receptor-mediated endocytosis - Specific and usually unique to cell function - Cell surface receptors recognize high-affinity ligands - Clustering of receptors allows selective concentration of endocytic vesicles without engulfing large amounts of extracellular fluid (as in pinocytosis) - Can involve the recruitment of cytosolic clathrin, forming a clathrin-coated pit, which is then internalized - Depending on cell-type and ligand, vesicle can have multiple fates including fusion with endosomes or lysosomes - Receptor is often recycled back to the cell surface Vesicular transport across membranes: endocytosis - There are three common types of endocytosis: 1) Pinocytosis 2) Phagocytosis 3) Receptor-mediated endocytosis → potocytosis - Restricted to small molecules (e.g. vitamins) - Generates relatively small vesicles called caveolae - Contents delivered to the cytosol (rather than lysosomes, etc) Vesicular transport is a specialized mechanism to transport molecules across membranes - Does not require molecules to pass through membrane – transport is achieved by enclosure into vesicles that pinch off from the membrane 1) Endocytosis: membrane envaginations enclose small volume of extracellular fluid, which are taken into the cell 2) Exocytosis: intracellular membrane-bound vesicles fuse with the plasma membrane and release their contents into the extracellular fluid http://starklab.slu.edu/neuro/Synapses.htm Vesicular transport across membranes: exocytosis - Two functions: 1) Replaces cell surface membrane lost during endocytosis 2) Allows secretion of membrane impermeable molecules into extracellular fluid - Allows for build up of material stored in secretory vesicles and made available for rapid secretion when signalled to do so (e.g. neurotransmitter or hormone release) Vander, Fig. 6.33 Outline: molecules can cross membranes through multiple mechanisms 1. Diffusion 2. Protein-mediated transport 3. Vesicular transport 4. Epithelial transport Epithelial cells line the cavities and surfaces of vessels and organs - Surface facing hollow organ/tube called apical membrane - Opposite surface (typically adjacent to blood vessels) called basolateral membrane - Two possible pathways across epithelial membrane: 1) Paracellular pathway (between cells) 2) Transcellular pathway (through cell) Epithelial cells line the cavities and surfaces of vessels and organs - Surface facing hollow organ/tube called apical membrane - Opposite surface (typically adjacent to blood vessels) called basolateral membrane - Two possible pathways across epithelial membrane: 1) Paracellular pathway (between cells) Movement limited by presence of tight junctions, limited to diffusion of water and small ions Epithelial cells line the cavities and surfaces of vessels and organs - Surface facing hollow organ/tube called apical membrane - Opposite surface (typically adjacent to blood vessels) called basolateral membrane - Two possible pathways across epithelial membrane: 2) Transcellular pathway (through cell) Utilizes processes of diffusion and protein-mediated transport just described Often involves flow against a concentration gradient, requiring ATP Epithelial cells line the cavities and surfaces of vessels and organs - Surface facing hollow organ/tube called apical membrane - Opposite surface (typically adjacent to blood vessels) called basolateral membrane - Two possible pathways across epithelial membrane: 2) Transcellular pathway (through cell) Utilizes processes of diffusion and protein-mediated transport just described Often involves flow against a concentration gradient, requiring ATP Outline: molecules can cross membranes through multiple mechanisms 1. Diffusion 2. Protein-mediated transport 3. Vesicular transport 4. Epithelial transport Learning outcomes At the end of Part 2, you should be able to identify and describe the different processes of membrane transport: a. Diffusion i. Understand the contribution of chemical, electrical, and electrochemical driving forces in the process of diffusion through membranes b. Protein-mediated transport i. Understand the contribution of proteins to membrane transport, including facilitated diffusion and active transport c. Vesicular transport i. Describe endocytosis and distinguish between pinocytosis, phagocytosis, and receptor-mediated endocytosis ii. Describe exocytosis d. epithelial transport i. Identify and define the transcellular and paracellular pathways used to cross layers of epithelial cells Physiology 210 Cell Physiology Part 1: Cellular Structure Part 2: Membrane transport Part 3: Cellular signaling Overview: Physiological homeostasis depends on communication between cells Focus today is on how cells receive signals and process them to generate a response Introduction: Signaling stimulus receptor response Different signals generate different response Introduction: Examples of signals that generate a physiological response… stimulus receptor response Patellar reflex: https://faculty.washington.edu/chudler/chreflex.html (8/2018) Pupil dilation: https://giphy.com/gifs/love-eyes-new-Qp9No07RFWfZu (8/2018) Milk ejection reflex: https://www.slideshare.net/hanasheque/breastfeeding-14847728 (8/2018) Introduction: physiological homeostasis depends on cell communication STIMULUS RELEASE OF SIGNALLING MOLECULE signal SIGNAL RECEIVED BY TARGET CELL receptor TARGET CELL RESPONSE Outline: Physiological homeostasis depends on communication between cells How do cells communicate with each other (intercellular communication)? 1) Receptors How do cells process signals (intracellular communication)? Once a cell has received a signal, it must be “processed”, this is called signal transduction 2) Lipid-soluble messengers 3) Water-soluble messengers → second messengers Intercellular signals are received by receptors Cells receive signals from other cells via receptor proteins → Depending on the signal, receptors can be located on the outside of the cell, or inside it Water-soluble signal lipid-soluble signal X - Most common - Bind to plasma transmembrane receptors - Can diffuse through membrane - Bind to intracellular receptors - Generally transduce signal via change in gene expression Ligand-receptor interactions dictate cellular signaling - Cell signaling depends on ligand-receptor interactions, including: 1) Specificity 2) Affinity 3) Saturation 4) Competition Ligand-receptor interactions dictate cellular signaling - Cell signaling depends on ligand-receptor interactions, including: 1) Specificity - The ability of a receptor to only bind a limited number of ligands Only Cell A can receive this signal, because it is the only one that expresses the specific receptor Vander, Figure 5.2 Ligand-receptor interactions dictate cellular signaling - Cell signaling depends on ligand-receptor interactions, including: 1) Specificity 2) Affinity – Strength of ligand binding to receptor 3) Saturation – Extent to which receptors are bound by ligand (100% = fully saturated) 4) Competition – Presence of other ligands which “compete” for receptor binding sites Vander, Figure 5.3 Competition between ligands is the molecular basis for many drugs - Cell signaling depends on ligand-receptor interactions, including: 4) Competition – Presence of other ligands which “compete” for receptor binding sites endogenous ligand agonist antagonist X no effect effect effect Fentanyl Respiratory depression Naloxone Opiod receptor Intercellular signalling can be regulated at the receptor level - Primary way to regulate receptors is through their number - Down-regulation - a lowering of the number of target cell receptors - Can occur in response to sustained high levels of signal (negative feedback) - Reduces cell response to frequent/intense stimulation - Common mechanism to down-regulate plasma membrane receptors is through internalization (includes receptor-mediated endocytosis) Intercellular signalling can be regulated at the receptor level - Primary way to regulate receptors is through their number - Down-regulation - a lowering of the number of target cell receptors - Can occur in response to sustained high levels of signal (negative feedback) - Reduces cell response to frequent/intense stimulation - Common mechanism to down-regulate plasma membrane receptors is through internalization (includes receptor-mediated endocytosis) - Up-regulation - An increase in the number of target cell receptors - Can occur in response to sustained low levels of signal (positive feedback) - Increases cell response to low-level stimulation - Can occur through increased insertion of receptor-containing vesicles into the cell membrane The process of signal transduction translates a signal into a cellular response Ligand binding Receptor activation to receptor (conformation change) signal transduction Cellular response: 1) Change in membrane properties 2) Cellular metabolism 3) Secretory activity 4) Rate of proliferation/differentiation Signal transduction pathway depends 5) Contractility or other activity on signal and receptor location Transduction of lipid-soluble signals - Examples include steroid hormones, thyroid hormone, and vitamin A - Response primarily mediated by nuclear receptors, leading to change in gene expression 1) Circulating signal diffuses from circulation across membrane into cell 2) Signal enters nucleus and binds receptor* 3) ligand-receptor complex functions as a transcription factor, changing expression (mRNA) level of target gene 4) Change in mRNA abundance effects change in protein level, leading to cellular response Vander, Figure 5.4 Transduction of water-soluble signals - More complex! - Examples include polypeptide hormones (e.g. insulin) and neurotransmitters - Signal transduction occurs in two phases: 1) binding of signal to receptor (first messenger) 2) signals generated by receptor activation (second messenger) - Many second messenger systems relying on phosphorylating proteins to effect change - Protein phosphorylation changes its structure, eliciting a response (can be activation or inhibition) - Enzymes that phosphorylate proteins are called protein kinases - Phosphorylation typically occurs at tyrosine reside, therefore called receptor tyrosine kinases - Multistep pathways can be complex... Transduction of water-soluble signals No need to memorize! MAP Kinase MAP Kinase Kinase MAP Kinase Kinase Kinase … Second messenger systems can create cascades that amplify a signal This system allows signalling molecules at low extracellular concentrations to have large effects e.g., one molecule of epinephrine can stimulate the liver to generate and release 108 molecules of glucose Vander, Figure 5.8 Transduction of water-soluble signals - Common mechanisms of water-soluble ligand signaling 1) Receptors that function as ion channels 2) Receptors that function as enzymes 3) Receptors that interact with cytoplasmic kinases (called janus kinases) 4) Receptors that interact with G-proteins (aka g-protein coupled receptors) Transduction of water-soluble signals - Common mechanisms of water-soluble ligand signaling 1) Receptors that function as ion channels Vander, Figure 5.5 “Ligand-gated ion channel” Transduction of water-soluble signals - Common mechanisms of water-soluble ligand signaling 2) Receptors that function as enzymes Vander, Figure 5.5 Transduction of water-soluble signals - Common mechanisms of water-soluble ligand signaling 3) Receptors that interact with cytoplasmic kinases (called janus kinases) - Membrane receptor has no intrinsic enzyme activity, but upon activation of the receptor, an associated kinase will be turned on Vander, Figure 5.5 Transduction of water-soluble signals - Common mechanisms of water-soluble ligand signaling 4) Receptors that interact with G-proteins (aka g-protein coupled receptors) - Largest group of receptors for water-soluble signals - G-proteins “couple” receptor with effector proteins to generate second messengers Vander, Figure 5.5 G-proteins are complex mediators of signaling through G-protein coupled receptors - G proteins are made up of three subunits - Alpha subunit: binds GDP/GTP - Beta-gamma subunit complex: help anchor alpha subunit in membrane - Ligand binding changes affinity for alpha subunit to GTP away from GDP - GTP-GDP exchange causes dissociation between alpha subunit and beta-gamma complex - Activated alpha subunit binds to effector protein to initiate cellular response Vander, Figure 5.5 Different families of G-proteins elicit different cellular responses - Three major families of G-proteins exist: - Defined by second messenger system they modulate and how - Gi >>> inhibits production of cyclicAMP - Gs >>> activates production of cyclicAMP - Gq >>> activates phospholipase C Different families of G-proteins elicit different cellular responses - Three major families of G-proteins exist: - Defined by second messenger system they modulate and how - Gs >>> activates production of cyclicAMP Gi proteins work in the opposite way, but inhibit adenylyl cyclase cAMP is a potent second messenger, the levels of which are tightly regulated - cAMP acts by activating cAMP-dependent protein kinase (aka protein kinase A; PKA) - PKA has multiple targets, meaning it can elicit multiple responses in the same cell (and different response in different cells, depending on the suite of target proteins expressed) Vander, Figure 5.9 cAMP is a potent second messenger, the levels of which are tightly regulated - Because it is so potent, cellular levels of cAMP are tightly regulated - Synthesis of cAMP from ATP is catalyzed by adenylyl cyclase - Breakdown of cAMP is catalyzed by cAMP phosphodiesterase Gs proteins Gi proteins Vander, Figure 5.7 Different families of G-proteins elicit different cellular responses - Three major families of G-proteins exist: - Defined by second messenger system they modulate and how - Gi >>> inhibits production of cyclicAMP - Gs >>> activates production of cyclicAMP - Gq >>> activates phospholipase C PIP2 → IP3 + DAG Calcium as a second messenger 1. How do signals cause the cytosolic Ca2+ concentration to increase? 2. How does increased Ca2+ concentration elicit a cellular response? http://newsroom.cumc.columbia.edu/blog/2015/04/07/cellular-defect-linked-diabetes/ Calcium as a second messenger 1. How do signals cause the cytosolic Ca2+ concentration to increase? 2. How does increased Ca2+ concentration elicit a cellular response? - Activation of plasma membrane Ca2+ channel (This could be by signal binding as shown, or also via a change in membrane potential in the case of a voltage-gated Ca2+ channel) - Opening of Ca2+ channel on the ER membrane - Active transport of Ca2+ out of the cell blocked by a second messenger (not shown) Calcium as a second messenger 1. How do signals cause the cytosolic Ca2+ concentration to increase? 2. How does increased Ca2+ concentration elicit a cellular response? Typically via binding to proteins and activating them, e.g. Calmodulin Cessation of signal transduction pathways - All good things must come to an end… - Cessation of intracellular signaling is required to prevent overstimulation of the cell - Most commonly occurs at the level of receptor activation - decreased concentration of signal (breakdown/uptake/diffusion) - Change in receptor conformation (e.g. via phosphorylation) > changes signal’s binding affinity > prevents further G-protein binding to the receptor - Receptor mediated endocytosis Cessation of signal transduction pathways - All good things must come to an end… - Cessation of intracellular signaling is required to prevent overstimulation of the cell Receptor desensitization Learning outcomes At the end of Part 3, you should be able to identify and describe the structural components of cells: 1. Identify and describe the different processes contributing to cellular signaling a. Define the different types of receptors and understand how they are regulated b. Define the terms signal transduction, first messenger, and second messenger c. Understand the processes underlying signal transduction d. Understand the difference between lipid-soluble and water-soluble messengers, and the location of their respective receptors i. Describe how lipid-soluble chemical messengers exert their effects ii. Describe how water-soluble chemical messengers exert their effects 1. Identify and describe the function of channel-linked receptors, enzyme-linked receptors, and G-protein coupled receptors. 2. Identify the major second messenger systems and their function, including cyclic AMP, Inositol triphosphate/Diacylgycerol, and calcium e. Understand the process underlying the cessation of cellular signalling