Transduction in Endocrinology 317319 Department of Biology University

Transduction in Endocrinology 317/319 Department of Biology University of Massachusetts Boston Kenneth L. Campbell Assembled August 2007

What are transducers? Transducers are proteins that convert the information in hormonal signals into chemical signals understood by cellular machinery. They change their shape & activity when they interact directly with protein-hormone complexes. Usually enzymes or nucleotide binding proteins, they produce 2 nd messengers, or change the activity of other proteins by covalently modifying them (adding or removing phosphate, lipid groups, acetate, or methyl groups), or they interact with other proteins that do these things. They begin amplifying the energy content of the original hormone signals.

What are effectors? Effectors are the enzymes & other proteins that convert the transduced hormonal signal into biochemical changes that generate the cellular response to hormone binding. Usually amplify the signal further & allow cellular work to be done: cell motion, growth, division, altered metabolism, secretion, depolarization, etc.

Transduction: The biochemical mechanism(s) that allow the transfer of information between an occupied hormone-receptor & the molecules within the cell that result in production of a cellular response. Ligand Receptor Transducer Amplifiers Effector Change in metabolism Change in transcription/gene-read out Change in secondary hormone production

Transduction System Concepts Features of transduction that both alter protein shape & function Allosteric changes Phosphorylation Membrane Receptors usually for proteins & charged molecules rapid response systems, sec-min Intranuclear Receptors lipids & hydrophobic hormones longer term responses, min-days

Transduction Pathways Depend on Receptor Types Ion Channels Intracellular/Intranuclear Receptor Steroids (sex, adrenal, vitamin D, sterols) Thyronines (tri-iodothyronine) G-Protein Receptors/Serpentine Receptors c. GMP/NOS c. AMP/PKA/CREB PLC /PKC/Calcium ion Cytokine & GH Receptors JAK/STAT Tyr. K Ras/GAP/MEK/MAPK RAC/Rho PI 3 K PLC /PKC Cross-talk allows unique responses in specific tissues &/or at specific times.

Transduction Systems I: • Translate information in hormone messages into language that can be interpreted & acted upon by target cells. • For proteins, peptides, & hormones with a high ionic charge at neutral p. H, receptors are usually integral membrane proteins in the cell surface. When hormones bind, the receptors interact with membrane-bound or intracellular transducer proteins to begin the cascade of events leading to cellular response. • Some membrane receptors, e. g. , the acetyl-choline receptor, act as ion channels that open or close in response to hormone binding & induce changes via changes of the intracellular ion/charge balance. • For many lipophilic hormones, e. g. , steroids or thyronines, receptors are intracellular, usually intranuclear, proteins. When their specific ligands bind, the hormone-receptor complexes undergo conformational changes that allow them to interact with specific hormone recognition sites (HREs) in the DNA of the regulatory regions of certain genes.

Transduction paths involve allosteric changes to proteins &/or chemical modifications such as phosphorylation, methylation, or acetylation. These change transducer & effector molecule charges, shapes & functions. They often alter intracellular location & associations. The paths may yield rapid metabolic changes – often via membrane receptors -- or trigger longer term responses via altered transcription & translation of proteins – via intracellular/nuclear receptors.

Transduction Systems II: • Transduction processes involve allosteric changes in receptor &/or transducer protein shape. Signaling cascades of membrane-bound receptors almost always involve protein phosphorylation by kinases; kinases may be activated initially via generation of secondary messengers produced by allosteric activation of enzymes like adenylyl or guanylyl cyclase or via unmasking of kinase activities that are part of the cytoplasmic portions of the receptor proteins themselves. • Both allosteric changes &/or phosphorylation, which changes protein charge, alter protein shape &/or intercellular location & protein function. These are exactly the changes that trigger a biochemical & cellular response. This may occur without intervention of protein synthesis & therefore may be very rapid, milliseconds to minutes. • Opening or closing of ion channels also yields rapid responses either directly or via the intervention of phosphorylation cascades with the associated changes in protein functions. • Intranuclear receptor transduction also involves phosphorylation & allosteric changes. It normally triggers changes in gene transcription & subsequent protein production & frequently modulates changes during a longer time course of minutes to days.

How many kinds of transducers are there?

How Significant Are Transduction Systems? At least 16. 4%, or 1/6 of the genome is devoted to coding for proteins that are part of transduction systems. If many transcription factors & cell adhesion proteins are included, the fraction rises to ~25% -- fully one quarter of the genome’s entire Good site on transduction coded content! l V http: //employees. csbsju. edu/hjakubowski/classes/ch 331/signaltrans/olsignalkinases. html

Receptor-binding specificity of VEGF family members & VEGFR-2 signaling pathways Note that some hormones may act via multiple receptors depending on hormonal concentration & receptor numbers. Conversely, some receptors may respond to several related hormones, again, depending on hormone concentration & receptor numbers. The activated pathways may trigger similar or dissimilar events & may act independently, synergistically, or antagonistically. Flexible cellular response! Hiroyuki Takahashi, Masabumi Shibuya, The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions, Clin. Sci. (2005) 109, 227 -241 Clinical Science www. clinsci. org

Can single cells make or sense more than one hormone at a time? Yes, cells can make multiple hormones, even of differing chemical classes, & they can sense multiple signals -- & integrate them -- all at once. Examples: Ovarian granulosa cells make inhibin (protein), estradiol (steroid), & androstenedione (steroid) during the follicular phase of the ovarian cycle. At the same time they respond to FSH & growth factors (proteins), estradiol (steroid), & thyroxine (amino acid derivative), along with other hormones. Anterior pituitary gonadotropes respond to LHRH (peptide) & inhibin (protein), estradiol, testosterone, progesterone, & glucocorticoids (steroids) while they make both FSH & LH (proteins).

The next two slides present links to extensive descriptions of the known transduction pathways &/or to details on particular steps in each transduction path. Jakubowski – Chapter 9 – Signal Transduction http: //employees. csbsju. edu/hjakubowski/classes/ch 331/sig naltrans/olsignalkinases. html

path. Finder is a program which finds signal transduction pathways between first, second, or nth messengers and targets within the cell. The usefulness of path. Finder consists in its ability to identify all possible signal transduction pathways connecting any starting component and target for a given set of possible two-component pathways in the path. Finder database. At present, there are 60 such two-step pathways in the path. Finder db. Addition of two-step pathways is ongoing. path. Finder can also identify all pathways connecting a starting component and a target when one or more intermediate pathway components are removed (excluded). This allows investigation of minimal activation sets and predicts effects of inhibitors of specific pathways on target activation from a given nth intracellular messenger. path. Finder is not quantitative: partial inhibitions and partial activations are not within the purview of path. Finder. Combinatorial activations, however, can be analyzed using path. Finder, although these are limited in the present version. Please send your comments or requests for on-line tutorials on path. Finder to eidenl@mail. nih. gov. path. Finder was conceived and constructed as a collaboration between Molecular Science Institute's Larry Lok and Lee Eiden of the NIMH-IRP, and has been adapted for Web use by Margaret Dayhoff-Brannigan, NIMH-IRP summer intern. Link to path. Finder Link to view CCList intramural. nimh. nih. gov/lcmr/smn/

Basic Schematic of Major Signaling Pathways homepages. strath. ac. uk/. . . /BB 329/MCSlect 6. html

More Detailed Schematic of Major Membrane Receptor Transduction Pathways for G-Protein Receptors & for Receptors Triggering Tyrosine Kinase Activity http: //www. ust. hk/~stn/graphs/multiprotein_signal_transduction. html

Interacting pathways including those involved in cell-cell or cell-matrix interactions. http: //www. benbest. com/health/cancer. html

Another illustration of the overlaps & potential extent of intracellular cross-talk mediated by the common transduction pathways.

Discussion of Specific Transduction Pathways

c. GMP & Protein Kinase G

Glyceride, Phosphatide & Inositide Nomenclature & Relationships

Sperm binding to an egg triggers a transduction cascade involving phospholipase C, protein kinase C & a rise in intracellular Ca++ that acts as a wave passing across the egg cytoplasm beginning at the point of binding of the sperm. This can be watched by first injecting the egg with a calcium-sensitive fluorescent dye like green dextran. Under a fluorescence microscope, the calcium-bound dye appears as a red wave. http: //Whitaker 1996 Contr ol of Meiotic Arrest. htm

© 1997 Kenneth L. Campbell Note that in the small G protein pathways the kinases require accessory proteins to exchange GTP for GDP or to cleave GTP to GDP, that many of the proteins interact via sites involving phosphorylated tyrosine residues, & that there are often multiple steps of kinase action each of which amplifies the original hormonal signal.

Tyrosine kinase-activated networks often use the P~Tyr sites as binding domains where auxiliary proteins involved in the subsequent cascade events may dock, be allosterically modified & undergo functional alteration. SH 2 & SH 3, src homology domains 2 & 3, are the most common of the docking sites generated by tyrosine SH 2 domains are common protein motifs that phosphorylation by tyrosine kinase. can be found in many different tyrosinekinase mediated signal transduction pathways. They bind & recognize specific peptide sequences containing phoshorylated tyrosines (see figure). ccs. chem. ucl. ac. uk/research/sh 2. shtml

Schematic of the grb 2, sos, raf, mek, map. K, myc, jun/fos (AP-1) pathway leading from an extracellular growth factor signal to a change in gene expression.

Zbigniew Walaszek, Margaret Hanausek, Thomas J. Slaga, Combined natural source inhibitors in skin cancer prevention, Cellscience Reviews 1(3), ISSN 1742 -8130.

Many of the transducer proteins are found in multiple forms each of which is specific to a particular tissue or cellular function. 3 D Structures of GTPases in Action: http: //www. cs. stedwards. edu/chem/Chemistry/CHEM 43/GTP/Index. htm

Phosphoinositide-3 Kinase (PI 3 K) plays an important role in paths associated with cell fate. http: //www. benbest. com/health/cancer. html

Transcriptional Mechanism of Steroids http: //www. aw-bc. com/mathews/ch 23/fi 23 p 16. gif

Mechanism of T 3 4 functional intranuclear T 3 receptors: α 1, β 1, 2, 3; & 1 nonfunctional receptor, α 2. Expression varies with tissue & developmental stage. http: //www. addison. ac. uk/endocrine_modules/module 1/lecturers_ material/html_files/END 1. 08/index. htm

Cross your eyes, relax, & see if you can see how 2 molecules of steroid receptor, green & yellow, interact with a specific sequence, SRE, in DNA. Receptors for steroids, T 3, retinoids, vitamin D, & aryl hydrocarbons all work this way.

Steroid Receptors Bind HREs as Homo - or Heterodimers http: //gibk 26. bse. kyutech. ac. jp/jouhou/image/dna-protein/all/small_S 1 glu. gif

Receptor Binding to HREs Can Bend DNA & Alter Transcription http: //gibk 26. bse. kyutech. ac. jp/jouhou/image/dna-protein/all/small_S 1 run. gif

Biochemistry of Metabolism Signal Transduction Copyright © 1999 -2006 by Joyce J. Diwan. All rights reserved. The following slides can be found and downloaded at: http: //www. rpi. edu/dept/bcbp/molbiochem/MBWeb/mb 1/part 2/signals. htm

Many enzymes are regulated by covalent attachment of phosphate, in ester linkage, to the side-chain hydroxyl group of a particular amino acid residue (serine, threonine, or tyrosine).

w A protein kinase transfers the terminal phosphate of ATP to a hydroxyl group on a protein. w A protein phosphatase catalyzes removal of the Pi by hydrolysis.

Phosphorylation may directly alter activity of an enzyme, e. g. , by promoting a conformational change. Alternatively, altered activity may result from binding another protein that specifically recognizes a phosphorylated domain. w E. g. , 14 -3 -3 proteins bind to domains that include phosphorylated Ser or Thr in the sequence RXXX[p. S/p. T]XP, where X can be different amino acids. w Binding to 14 -3 -3 is a mechanism by which some proteins (e. g. , transcription factors) may be retained in the cytosol, & prevented from entering the nucleus.

Protein kinases and phosphatases are themselves regulated by complex signal cascades. For example: w Some protein kinases are activated by Ca++calmodulin. w Protein Kinase A is activated by cyclic-AMP (c. AMP).

Adenylate Cyclase (Adenylyl Cyclase) catalyzes: ATP c. AMP + PPi Binding of certain hormones (e. g. , epinephrine) to the outer surface of a cell activates Adenylate Cyclase to form c. AMP within the cell. Cyclic AMP is thus considered to be a second messenger.

Phosphodiesterase enzymes catalyze: c. AMP + H 2 O AMP The phosphodiesterase that cleaves c. AMP is activated by phosphorylation catalyzed by Protein Kinase A. Thus c. AMP stimulates its own degradation, leading to rapid turnoff of a c. AMP signal.

Protein Kinase A (c. AMP-Dependent Protein Kinase) transfers Pi from ATP to OH of a Ser or Thr in a particular 5 -amino acid sequence. Protein Kinase A in the resting state is a complex of: • 2 catalytic subunits (C) • 2 regulatory subunits (R). R 2 C 2

R 2 C 2 Each regulatory subunit (R) of Protein Kinase A contains a pseudosubstrate sequence, like the substrate domain of a target protein but with Ala substituting for the Ser/Thr. The pseudosubstrate domain of (R), which lacks a hydroxyl that can be phosphorylated, binds to the active site of (C), blocking its activity.

R 2 C 2 + 4 c. AMP R 2 c. AMP 4 + 2 C When each (R) binds 2 c. AMP, a conformational change causes (R) to release (C). The catalytic subunits can then catalyze phosphorylation of Ser or Thr on target proteins. PKIs, Protein Kinase Inhibitors, modulate activity of the catalytic subunits (C).

G Protein Signal Cascade Most signal molecules targeted to a cell bind at the cell surface to receptors embedded in the plasma membrane. Only signal molecules able to cross the plasma membrane (e. g. , steroid hormones) interact with intracellular receptors. A large family of cell surface receptors have a common structural motif, 7 transmembrane -helices. Rhodopsin was the 1 st member of this family to have its 7 -helix structure confirmed by X-ray crystallography.

w Rhodopsin is unique in that it senses light. w Most 7 -helix receptors have domains facing the extracellular side of the plasma membrane that recognize & bind particular signal molecules (ligands). The signal is passed from a 7 -helix receptor to an intracellular G-protein. w Seven-helix receptors are thus called GPCR, or G-Protein-Coupled Receptors. w Approximately 800 different GPCRs are encoded in the human genome.

G-protein-Coupled Receptors may dimerize or form oligomeric complexes within the membrane. Ligand binding may promote oligomerization, which may in turn affect activity of the receptor. Various GPCR-interacting proteins (GIPs) modulate receptor function. Effects of GIPs may include: w altered ligand affinity w receptor dimerization or oligomerization w control of receptor localization, including transfer to or removal from the plasma membrane w promoting close association with other signal proteins

w G-proteins are heterotrimeric, with 3 subunits , , . w A G-protein that activates cyclic-AMP formation within a cell is called a stimulatory G-protein, designated Gs with alpha subunit Gs. w Gs is activated, e. g. , by receptors for the hormones epinephrine and glucagon. The -adrenergic receptor is the GPCR for epinephrine.

The subunit of a G-protein (G ) binds GTP, & can hydrolyze it to GDP + Pi. & subunits have covalently attached lipid anchors that bind a G-protein to the plasma membrane cytosolic surface. Adenylate Cyclase (AC) is a transmembrane protein, with cytosolic domains forming the catalytic site.

The complex of & subunits G , inhibits G. The sequence of events by which a hormone activates c. AMP signaling: 1. Initially G has bound GDP, and , , & subunits are complexed together.

2. Hormone binding to a 7 -helix receptor (GPCR) causes a conformational change in the receptor that is transmitted to the G protein. The nucleotide-binding site on G becomes more accessible to the cytosol, where [GTP] > [GDP]. G releases GDP & binds GTP (GDP-GTP exchange).

3. Substitution of GTP for GDP causes another conformational change in G. G -GTP dissociates from the inhibitory complex & can now bind to and activate Adenylate Cyclase.

4. Adenylate Cyclase, activated by the stimulatory G -GTP, catalyzes synthesis of c. AMP. 5. Protein Kinase A (c. AMP Dependent Protein Kinase) catalyzes phosphorylation of various cellular proteins, altering their activity.

Turn off of the signal: 1. G hydrolyzes GTP to GDP + Pi. (GTPase). The presence of GDP on G causes it to rebind to the inhibitory complex. Adenylate Cyclase is no longer activated. 2. Phosphodiesterase catalyzes hydrolysis of c. AMP.

Turn off of the signal (cont. ): 3. Receptor desensitization occurs. This process varies with the hormone. w Some receptors are phosphorylated via specific receptor kinases. w The phosphorylated receptor may then bind to a protein -arrestin, that promotes removal of the receptor from the membrane by clathrinmediated endocytosis. 4. Protein Phosphatase catalyzes removal by hydrolysis of phosphates that were attached to proteins via Protein Kinase A.

Signal amplification is an important feature of signal cascades: w One hormone molecule can lead to formation of many c. AMP molecules. w Each catalytic subunit of Protein Kinase A catalyzes phosphorylation of many proteins during the life-time of the c. AMP.

w The stimulatory Gs , when it binds GTP, activates Adenylate cyclase. w An inhibitory Gi , when it binds GTP, inhibits Adenylate cyclase. Different effectors & their receptors induce Gi to exchange GDP for GTP than those that activate Gs. In some cells, the complex of G , that is released when G binds GTP is itself an effector that binds to and activates other proteins.

w Cholera toxin catalyzes covalent modification of Gs. • ADP-ribose is transferred from NAD+ to an arginine residue at the GTPase active site of Gs. • ADP-ribosylation prevents GTP hydrolysis by Gs . • The stimulatory G-protein is permanently activated. w Pertussis toxin (whooping cough disease) catalyzes ADP-ribosylation at a cysteine residue of the inhibitory Gi , making it incapable of exchanging GDP for GTP. • The inhibitory pathway is blocked. w ADP-ribosylation is a general mechanism by which activity of many proteins is regulated, in eukaryotes (including mammals) as well as in prokaryotes.

ADP ribosylation

Structure of G proteins: The nucleotide binding site in G consists of loops that extend out from the edge of a 6 -stranded sheet. Three switch domains have been identified, that change position when GTP substitutes for GDP on G. These domains include residues adjacent to the terminal phosphate of GTP and/or the Mg++ associated with the two terminal phosphates.

GTP hydrolysis occurs by nucleophilic attack of a water molecule on the terminal phosphate of GTP. Switch domain II of G includes a conserved glutamine residue that helps to position the attacking water molecule adjacent to GTP at the active site.

The subunit of the heterotrimeric G Protein has a b-propeller structure, formed from multiple repeats of a sequence called the WD-repeat. The -propeller provides a stable structural support for residues that bind G.

The family of heterotrimeric G proteins includes also: w transducin, involved in sensing of light in the retina. w G-proteins involved in odorant sensing in olfactory neurons. There is a larger family of small GTP-binding switch proteins, related to G.

Small GTP-binding proteins include (roles indicated): w w w initiation & elongation factors (protein synthesis). Ras (growth factor signal cascades). Rab (vesicle targeting and fusion). ARF (forming vesicle coatomer coats). Ran (transport of proteins into & out of the nucleus). Rho (regulation of actin cytoskeleton) All GTP-binding proteins differ in conformation depending on whether GDP or GTP is present at their nucleotide binding site. Generally, GTP binding induces the active state.

Most GTP-binding proteins depend on helper proteins: GAPs, GTPase Activating Proteins, promote GTP hydrolysis. A GAP may provide an essential active site residue, while promoting the correct positioning of the glutamine residue of the switch II domain. Frequently a (+) charged arginine residue of a GAP inserts into the active site and helps to stabilize the transition state by interacting with (-) charged O atoms of the terminal phosphate of GTP during hydrolysis.

w G of a heterotrimeric G protein has innate capability for GTP hydrolysis. It has the essential arginine residue normally provided by a GAP for small GTP-binding proteins. w However, RGS proteins, which are negative regulators of G protein signaling, stimulate GTP hydrolysis by G.

GEFs, Guanine Nucleotide Exchange Factors, promote GDP/GTP exchange. w An activated receptor (GPCR) normally serves as GEF for a heterotrimeric G-protein. w Alternatively, AGS (Activator of G-protein Signaling) proteins may activate some heterotrimeric G-proteins, independent of a receptor. Some AGS proteins have GEF activity.

Phosphatidylinositol Signal Cascades Some hormones activate a signal cascade based on the membrane lipid phosphatidylinositol.

Kinases sequentially catalyze transfer of Pi from ATP to OH groups at positions 5 & 4 of the inositol ring, to yield phosphatidylinositol-4, 5 bisphosphate (PIP 2). PIP 2 is cleaved by the enzyme Phospholipase C.

Different isoforms of Phospholipase C have different regulatory domains, & thus respond to different signals. A G-protein, Gq activates one form of Phospholipase C. When a particular GPCR (receptor) is activated, GTP exchanges for GDP. Gq -GTP activates Phospholipase C. Ca++, which is required for activity of Phospholipase C, interacts with (-) charged residues & with Pi moieties of the phosphorylated inositol at the active site.

Cleavage of PIP 2, catalyzed by Phospholipase C, yields 2 second messengers: w inositol-1, 4, 5 -trisphosphate (IP 3) w diacylglycerol (DG). Diacylglycerol, with Ca++, activates Protein Kinase C, which catalyzes phosphorylation of several cellular proteins, altering their activity.

IP 3 activates Ca++-release channels in ER membranes. Ca++ stored in the ER is released to the cytosol, where it may bind calmodulin, or help activate Protein Kinase C. Signal turn-off includes removal of Ca++ from the cytosol via Ca++-ATPase pumps, & degradation of IP 3.

Sequential dephosphorylation of IP 3 by enzymecatalyzed hydrolysis yields inositol, a substrate for synthesis of PI. IP 3 may instead be phosphorylated via specific kinases, to IP 4, IP 5 or IP 6. Some of these have signal roles. E. g. , the IP 4 inositol-1, 3, 4, 5 -tetraphosphate in some cells stimulates Ca++ entry, perhaps by activating plasma membrane Ca++ channels.

The kinases that convert PI (phosphatidylinositol) to PIP 2 (PI-4, 5 -P 2) transfer Pi from ATP to OH at positions 4 & 5 of the inositol ring. PI 3 -Kinases instead catalyze phosphorylation of phosphatidylinositol at the 3 position of the inositol ring.

PI-3 -P, PI-3, 4 -P 2, PI-3, 4, 5 -P 3, and PI-4, 5 -P 2 have signaling roles. Head-groups of these transiently formed lipids are ligands for particular pleckstrin homology (PH) & FYVE protein domains that bind proteins to membrane surfaces. Other protein domains called MARKS are (+) charged, and their binding to (-) charged headgroups of lipids like PIP 2 is antagonized by Ca++.

Protein Kinase B (also called Akt) becomes activated when it is recruited from the cytosol to the plasma membrane surface by binding to products of PI-3 Kinase, e. g. , PI-3, 4, 5 -P 3. w Other kinases at the cytosolic surface of the plasma membrane then catalyze phosphorylation of Protein Kinase B, activating it. w Activated Protein Kinase B catalyzes phosphorylation of Ser or Thr residues of many proteins, with diverse effects on metabolism, cell growth, and apoptosis. w Downstream metabolic effects of Protein Kinase B include stimulation of glycogen synthesis, stimulation of glycolysis, and inhibition of gluconeogenesis.

Signal protein complexes: Signal cascades are often mediated by large "solid state" assemblies that may include receptors, effectors, and regulatory proteins, linked together in part by interactions with specialized scaffold proteins. Scaffold proteins often interact also with membrane constituents, elements of the cytoskeleton, and adaptors mediating recruitment into clathrin-coated vesicles. They improve efficiency of signal transfer, facilitate interactions among different signal pathways, and control localization of signal proteins within a cell.

Signal complexes are often associated with lipid raft domains of the plasma membrane. Scaffold proteins as well as signal proteins may be recruited from the cytosol to such membrane domains in part by w insertion of lipid anchors w interaction of pleckstrin homology or other lipidbinding domains with head-groups of transiently formed phosphatidylinositol derivatives, such as PIP 2 or PI-3 -P.

AKAPs (A-Kinase Anchoring Proteins) are scaffold proteins with multiple domains that bind to w regulatory subunits of Protein Kinase A w phosphorylated derivatives of phosphatidylinositol w various other signal proteins, such as: • G-protein-coupled receptors (GPCRs) • Other kinases such as Protein Kinase C • Protein phosphatases • Phosphodiesterases AKAPs localize hormone-initiated signal cascades within a cell, and coordinate activation of protein kinases as well as rapid turn-off of such signals.