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Cellular Physiology - Dr. Ferguson .docx

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Physiology 3140A
Donglin Bai

Cellular Physiology: Dr. Steve Ferguson Lecture 2: Steroid Receptor Structure Function General Characteristics Ligands • Small hydrophobic molecules that have very different structures and physiological functions • All act through similar mechanism Examples of Ligands: • Sex hormones o Produced from cholesterol o Made in testes and ovaries o Responsible for determining secondary sexual characteristics that distinguish males and females • Cortisol thyroid hormone o Produced from cholesterol o Cortisol is produced in the cortex of the adrenal glands o Influences cellular metabolism • Vitamin D o Produced from cholesterol o Produced in the skin in response to sunlight o Converted to its active form in the liver and kidneys o Regulates Ca2+ metabolism promoting uptake in the gut and reducing secretion from kidneys • Retinoid hormones o Made from vitamin A o Important local mediators in vertebrate development Receptors • Multiple intracellular receptors which are structurally-related and constitute an “intracellular receptor superfamily” • Receptor family contains 30 members o Including some “orphan” receptors for which ligands haven’t been found yet • Steroid hormones aren’t water soluble • Made soluble by extracellular fluids by binding to carrier proteins • Most water soluble hormones are broken down in the bloodstream in a matter of minutes • Steroid hormones persist for hours (sex hormones) to days (thyroid hormone) • Cellular mechanisms of action of steroids are related • Effector molecules circulate at nanomolar concentrations in blood and freely diffuse through cell membranes • Interaction of hormone with receptor converts the transcriptionally inactive receptor protein into a form that recognizes and binds to specific DNA sequences o Regulatory regions of target genes • Interactions lead to alterations in: o The rate of gene transcription  Both increased and decreased o Magnitude of which depends on the cellular promoter o Context of the bound receptor • Steroid receptors bind as protein dimers (receptor pairs) to specific gene Hormone Responsive Elements (HREs) Steroid Receptors • Found in the cytosol that translocate to the nucleus upon ligand binding • Can also be found bound to DNA in the nucleus even in the absence of agonist • Ones found in the nucleus are bound to inhibitory proteins o Ligand binding induces a conformational change that induces the release of the inhibitor complex and activation of receptor • Transport of newly synthesized receptor molecules into the nucleus requires nuclear localization signal • Steroid hormones are involved in: o Cell differentiation o Cellular homeostasis o Morphogenesis • Diseases linked to steroid receptors: o Cancer o Metabolic diseases o Reproductive diseases • Therapeutic drugs are based on hormone replacement or anti-steroid receptor antagonists Examples of Steroid Antagonists: • Tamoxifen o Anti-estrogen drug for treatment of breast cancer o Currently largest selling anti-cancer drug • RU486 o Abortion drug o Anti-progesterone also used in cancer treatment Receptor Superfamily Structure • Large diversity in molecular size of steroid receptors o 424 amino acids for the Vitamin D receptor o 984 amino acids for the aldosterone receptor • Alignment of primary sequences of receptors have revealed several highly conserved regions in the carboxyl-terminal half of the receptors • Considerable structural heterogeneity and sequence difference in the amino termini Structural Regions of the Receptors 1. Amino-terminal domain • Variable domain • This region varies considerably in size and amino acid composition from receptor to receptor • This area of the receptor is involved in transcriptional transactivation leading to modification of gene expression 2. DNA binding domain • Domain of steroid receptors that bind to specific DNA sequences in the “hormone response element” of genes • Region contains numerous basic amino acid residues and an invariant organization of nine cysteine residues • Eight of these cysteine’s bind two zinc molecules (4 cystenins per one zinc) o Forms two “zince-fingers” • These zinc-fingers align the ligand-bound receptor as a transcription factor in the major groove of the DNA 3. Hormone (ligand) binding domain • Located at carboxy terminus • Ligand binding domain overlaps with other functional domains required for: o Dimerization o Transcriptional actiation o Interaction with the heat shock proteins o Sequences required for nuclear localization Hormone Response Element (HRE) • DNA sequences required for the specific binding of nuclear receptors • Generally situated in the regulatory domains of target genes upstream of the transcription initiation site o Site that binds DNA polymerase • Contain two short (5-6bp) segments (half-sites) of a very similar or identical sequence that form the nucleotide base pair consensus sequence for steroid receptor binding • Binding of steroid receptors to these two nucleotide half-sites is “functional” o Leads to altered gene transcription • Binding the nuclear receptors to HRE sequences usually leads to enhancement of transcription o Can have opposite effect leading to inhibition of transcription Activation of Hormone Receptors • Biologically inactive, ligand-free receptors are localized predominantly in the cell nucleus o Not tightly bound to DNA • Ligand free receptors don’t bind to cognate hormone response elements o Might be due to association with heath-shock proteins (hsp) • There are certain receptors that exist within the cell as large oligomeric complexes that are associated with three different heat shock proteins o Hsp90 o Hsp70 o Hsp56 o These receptors include:  Estrogen  Progesterone  Glucocorticoid  Androgen  Aldosterone • Certain receptors don’t associate with heat shock proteins o Thyroid o Vitamin D o Retinoic acid receptors • Hsp90 negatively regulates receptor-DNA interactions • Binding of hormone to the ligand binding site results in conformational change in the receptor o Displaces the inhibitory heat shock proteins o Leads to transformation of receptor into transcriptionally active form • Ligand-induced transformation “activates” the binding to specific DNA consensus sequences o Hormone response elements Lecture 3: G-Protein Coupled Receptor Structure Function Activators of G-Protein Coupled Receptor Signaling • Receptors o G-protein coupled receptors (GPCRs) from the largest family of integral membrane receptor proteins in C. elegans – comprising 5% of genome & in humans just greater than 2% of genome o Estimated that more than 650 GPCRs exist and of this number ~300 encode olfactory receptors o Of the remaining only 250 receptors have been ascribed endogenous ligands leaving 100 orphan receptors with no known function – ½ of all prescribed drugs target GPCRs either directly or indirectly • Ligands o Enormous diversity in the ligands that activate GPCRs o Diverse: light, smell, taste, hormones, neurotransmitters, ions, nucleotides, small peptides, large glycoproteins, fatty acids, amino acids & pheromones o Same ligand can activate multiple different receptors in the same family o Ex.  9 distinct receptors are activated by adrenaline  5 receptors are activated by acetylcholine  7 receptors are activated by glutamate  15 receptors are activated by serotonin • GPCR Topology o 7 transmembrane spanning receptors that are also referred to as serpentine or heptahelical receptors o 7 transmembrane domains: composed of protein helices & are lined by intracellular & extracellular loop domains – 7 transmembrane domains form a hydrophobic pocket that forms the ligand binding pocket o Amino terminal domain of all GPCRs is extracellular & in combination with the extracellular loop domains: contributes to the binding of peptide ligands & large glycoprotein hormones o Carboxyl-terminal domain: intracellular & contains the sites for G-protein coupled receptor phosphorylation & contributes to binding of GPCR regulatory proteins such as arrestins o Intracellular loop domains as well as the carboxy-terminal tail contribute to G-protein coupling & association with GPCR regulatory proteins o Lot of molecular diversity between receptor subtypes  Some have large extracellular tails while some have relatively short extracellular tails  Some have large third intracellular loops whereas others have small intracellular loops  Some have long carboxy-terminal tails while others have short carboxy-terminal tails o Differences dictate the type of ligands, G proteins & regulatory proteins that will associate with the receptors & define molecular diversity of GPCR signaling • GPCR Agonist Binding Theory o Agonist binding to receptor facilitates ability of GPCR to adopt a molecular configuration that allows it to catalyze exchange of GDP for GTP on the α-subunit of the heterotrimeric G protein o Receptor can spontaneously adopt an active configuration even in the absence of agonist o Ability of GPCRs to activate G protein signaling in absence of agonist activation is referred to as spontaneous or intrinsic activity (inactiveR versus R* (activate) o Extend of spontaneous (intrinsic) activity differs from one receptor to the next o Ex. angiotensin receptor – less spontaneous than glutamate receptors o Ex. β-adrenergic receptors exhibit low intrinsic activity whereas metabotropic glutamate receptors (mGluRs) exhibit profound intrinsic receptor activity o Several mutations in amino acid sequences encoding GPCRs lead to increased spontaneous receptor activity that leads to diseases o Examples include: precocious puberty (LH hormone receptor overactivity), thyroid adenomas (TSH receptor overactivity) & retinitis pigmentosa (degeneration of retina due to overactivation of rhodopsin) • Pharmacological Definitions o Full agonists: stabilize an active receptor conformation that is able to maximally mediate receptor G-protein coupling (full R* conformation) o Partial agonists: stabilizes an active receptor conformation that is unable to maximize receptor G-protein coupling (less effective at stabilizing active state – partial activity) o Inverse agonists: are drugs that do not stimulate receptor activation but rather selectively recognize or stabilize the inactive receptor conformation & thereby reduce spontaneous or intrinsic activity of GPCRs (R conformation) o Full antagonist:  Do not preferentially recognize or stabilize either the inactive or active receptor conformation (does not select for R or R* state – doesn’t change activity of receptor)  Treatment of cells with full antagonists will result in no net change in receptor activity  Antagonist will block the activity of both agonist & inverse agonists • Ligand Binding (GPCR agonists bind to and activate GPCRs) o Constitutively bound ligand:  Rhodopsin the receptor for light is constitutively bound to a chromophore (retinal) within a binding pocket formed by 7 transmembrane helices  Activation of receptor by a photon a light involves isomerization of chromophore from 11- cis-retinal to all-trans-retinal causing a change in orientation & distribution of transmembrane helices  Cis (light: ) trans o Hydrophobic ligand binding pocket:  In the case of GPCRs that bind small molecules such as neurotransmitters & small peptide agonists & antagonists bind within a hydrophobic ligand pocket formed by 7 transmembrane spanning domains (most common) o Extracellular domain ligand binding:  In the case of peptide or large glycoprotein hormone receptor agonist binding involves the extracellular loop domains & the amino-terminal tail of the receptors o Venus-fly trap model:  Unlike observed for most receptors that bind small molecules, metabotropic glutamate receptor (mGluR) activation involves glutamate binding to a stretch of amino acids within the long extracellular amino-terminal tail  Structure of domain resembles bacterial periplasmic binding proteins  Binding of ligand = Venus fly-trap model o Protease-activated receptors (tethered-ligands):  Ex. thrombin receptor encode their own tethered ligands within amino terminal tail of the receptor that bind back into hydrophobic ligand binding pocket formed by transmembrane domains when revealed  Ligands are revealed following removal of the first 8 amino acids from amino terminus by proteases Heterotrimeric G Proteins • General characteristics o Comprised of α, β, γ subunits o βγ-subunits function as a complex – single functional component o Human genome encodes genes for 27 α subunits, 5 β subunits & 13 γ subunits o Some of these genes also have multiple protein products – alternative RNA splicing o Hydrophilic in nature but are localized at plasma membrane o α & γ subunits are post-translationally modified with addition of covalently bound fatty acids o For the α subunit  C-terminal is important for receptor & effector interactions  N-terminal is responsible for membrane anchorage, myristoylation & interaction with βγ • G-protein activation mechanism o G proteins functionally couple receptors to target enzymes or ion channels in plasma membrane o G proteins have intrinsic GTP-ase activity & can switch between an inactive (GDP bound) & active conformation (GTP bound) in response to receptor actndation o G proteins act as molecular switch – triggers generation of a 2 messenger cascade following GPCR ligand binding • Classification of heterotrimeric G-proteins o Classified by α subunit into 4 main classes: Gs, Gi, Gq 12G o Gs = stimulatory G protein - coupled to activation of adenyl cyclases & cAMP formation o Gi = inhibitory G protein, - copled to inactivation of adenyl cyclase & reduces/attenuates cAMP formation in response to activation of Gs coupled receptors o Gq = coupled to activation of phospholipase Cβ & formation of diacyglycerol (DAG) and inositol 1,4,5 phosphate (IP3) followed by release of calcium from intracellular stores o G 12coupled to activation of Rho GTPase via activation of p115 RhoGEF o Also classified by βγ-subunits – contribute to the regulation of GPCR signaling through their regulation of adenyl cyclases, ion channels (K channels) & PI3 kinases etc. • Tools to study G protein α subunits o Fluoride: functions as GTP-independent activator G-proteins o Non-hydrolyzable analogues of GTO: GTPγS produces prolonged G protein activation due to resistance to GTPase mediated hydrolysis of gamma phosphate group • Toxins o Cholera toxins  Severe diarrheal disease caused by bacterium Vibrio Cholerae – most rapidly fatal diseases (person may die within 2-3 if no treatment)  Transfers ADP-ribose moiety from NAD to a specific arginine residue within Gαs  Functions to suppress GTPase activity thereby locking G protein in an active GTP-bound conformation  Increase activity of adenylate cyclase by increased Gs activation o Pertussis Toxin:  Cause of whopping cough – mediated by bacterial infection by Bordetella pertussis  ADP-ribosylates Gαi protein at cysteine residue near C-terminal  Results in G protein uncoupling from receptor & complex remains bound to GDP and is thus unable to exert its functions  Inactivates Gi o Functional Roles of βγ-subunit  Facilitator • Negative regulator o Promotes inactive state of the G protein because βγ subunits increase affinity of α-subunit for GDP (100-fold) o Holds G protein in inactive state in the absence of agonist • Facilitates receptor dependent G-protein activation o βγ-subunits presents α-subunits in correct orientation for receptor activation & promotes association of GDP-bound α-subunit with ligand- bound receptor • Broader organizational role o Serving as sites for localizations of α-subunits to the inner surface of plasma membrane  Activator + • Activates K channels (I K,ACh o Either directly or indirectly through activation of phospholip2se A & generation of arachidonic acid • Activates phospholipase C o Leading to increase phosphoinositide hydrolysis • Regulates adenylate cyclase o βγ-dimers from activated Giassociate with & promote inactivation of free stimulatory Gαs • Modulates receptor function o Controls the location of receptor-specific kinases that blunt receptor activity by targeting certain kinases to enhance ligand-induced receptor phosphorylation & desensitization Lecture 4: G Protein-Coupled Receptor Structure Function Receptor Desensitization • Receptor inactivation: exposure of GPCRs to agonists often results in rapid attenuation of receptor responsiveness – process: desensitization – is achieved by 4 mechanisms Phosphorylation • Occurs on intracellular serine residues by protein kinases resulting in the inability of receptor to couple heterotrimeric G-protein & stimulate the exchange of GDP for GTP on α subunit • Represents the most rapid means by which GPCR desensitization can be achieved • Within seconds to minutes following ligand binding • Mediated by o 2 messenger dependent protein kinases (cAMP-dependent PKA & PKC)  Thought to represent the predominant mechanisms by which desensitization is achieved at low agonist concentrations  Provides feedback upon receptor to modulate the extend & duration of GPCR signaling  Activation occurs as consequence of receptor activation  Coupling of GPCRs via Gs to the activation of adenylyl cyclase leads to increased intracellular cAMP & activation of cAMP-dependent PKA  Coupling of receptors via Gq to activation of PLC-β & increased I3 & diacylglycerol formation results in activation of protein kinase C (PKC)  Examples: • β2-adrenergic receptor mediated activation of PKA & metabotropic glutamate receptor activation of protein kinase C • PKA phosphorylates the β2-adrenergic at 2 PKA consensus sites • One site is found in third intracellular loop & 2 is found in the proximal r egion of the C-terminal tail • Phosphorylation sites are localized to regions of the receptors that are essential for G protein coupling • Post-translational modification of the receptor at these sites by PKA- or PKC- mediated phosphorylation inhibits the ability of the receptor to associate with heterotrimeric G proteins o GPCR-specific kinases called G-protein coupled receptor kinases (GRKs) Internalization • GPCRs can be temporarily removed into the interior of the cell so that they are no longer available at the cell surface for ligand binding & are unable to interact with the heterotrimeric G protein Degradation • GPCRs are destroyed in lysosomes & proteasomes following internalization Synthesis Inhibition • Decrease in GPCR protein synthesis as a consequence of decreased mRNA synthesis due to 2 messenger-dependent inhibition in GPCR gene transcription • Ex. PKA phosphorylation of CREB decrease β2-adrenergic receptor mRNA synthesis • Extend of receptor desensitization varies from complete termination of signaling (visual & olfactory systems) to attenuation of agonist potency & maximal responsiveness (β2-adrenergic receptor) • Extend of receptor desensitization is regulated by a number of factors including: receptor structure & cellular environment (ex. compliment of kinases expressed by a particular cell) Role of Desensitization in Patterning of Signal Transduction nd • Activation of GPCRs generally results in generation of a single 2 messenger response • Activation of feedback phosphorylation by 2 messenger dependent protein kinases followed by receptor dephosphorylation by phosphatases can lead to repetitive activation of signaling responses: oscillations (activation/inactivation cycles of receptors and is associated pathway) • Ex. o Metabotropic glutamate receptors (mGluRs) are members of G protein-coupled receptor superfamily that are activated by excitatory glutamate amino acid o Play an essential role in regulating neural development & plasticity o Activation of mGluRs leads to increases in membrane bound diacylglycerol (DAG) & IP 3 concentrations, release of Ca from intracellular stores & activation of PKC isoforms o mGluR activation gives rise to repetitive increases in intracellular free Ca (oscilations) that are translated into repetitive of PKC & repetitive phosphorylation & dephosphorylation (by phosphatases) of the receptor • Role of Ca Oscillations o General  Fine tuning of inflammatory immune responses  Regulation of release of insulin from β-islet cells in pancreas  Sustained activation of mitochondrial activity  At different frequencies can activate different transcription factors  different outcomes • NFAT  activated by high frequency oscillations • NF K  activated by low frequency oscillations o Central nervous system  Increased activation of calmodulin kinase II activity  Regulation of neurotransmitter & neurotransmitter channel expression  Regulation of synapse formation & elimination in developing CNS  Differentiation & migration of neuronal stem cells  Regulation of memory & learning Lecture 5: G Protein-Coupled Receptor Regulation Homologous vs. Heterologous Desensitization • 2 messenger-dependent protein kinases exhibit the capacity to phosphorylate both agonist-activated receptors & receptors that have not been exposed to agonist • Heterologous receptor desensitization o Agonist-activation independent phosphorylation (inactivation) of GPCRs (phosphorylates that which has not been agonist-activated) o Occurs when 1 receptor system (ex. β2-adrenergic receptor) contributes to desensitization of another receptor system that has not been agonist activated (ex. D1 dopamine receptor) o Post-translation modification of the 2 receptor due to PKA- or PKC-mediated phosphorylation inhibits ability of this receptor to associate with heterotrimeric G proteins in response to subsequent activation by its agonist • Homologous receptor desensitization o Involves desensitization of receptors that have been agoni
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