Chapter 11 - Biochem
Acetylcholine

Nicotinic acetylcholine receptors – receptors on PM of skeletal muscle cells where ACh attaches – receptor subunits assembled around channel that has funnel-shaped opening in center o ACh attachment causes conformational change to open narrow portion of the channel, allowing Na + to diffuse in and K + to diffuse out o Once ACh secretion stops, message is rapidly terminated by acetylcholinesterase (enzyme located on postsynaptic membrane that cleaves ACh) – also terminated by diffusion of ACh away from the synapse

ACh – nicotinic receptors and muscarinic receptors o Curare is inhibitor of nicotinic receptors o Atropine is inhibitor of muscarinic receptors – used when acetylcholinesterase has been inactivated by
 nerve gases or chemicals such that atropine will block effects of excess ACh present at synapse
M2 muscarinic receptors activate Gα i/0
heterotrimeric G protein in which release of βγ-subunit controls K + channels and pacemaker activity of heart o Epinephrine has several types of heptahelical receptors
 Β-adrenergic receptors work through Gα s
and stimulate adenylyl cyclase
 α
2
-adrenergic receptors work through Gα i
protein and inhibit adenylyl cyclase
 α
1
-adrenergic recptors work through Gα q
-subunits and activate phospholipase C
β
Types of Messengers

Nervous system uses biogenic amines and neuropeptides for messengers

Immune system uses cytokines for messengers (regulate responses designed to kill invading microorganisms) o Chemokines – chemotactic cytokines that have ability to induce movement in targeted cells toward source of chemokines

Eicosanoids – prostaglandins, leukotrienes, thromboxanes – control cellular function in response to injury – derived from arachidonic acid o Present in almost every cell, but different cells produce different eicosanoids o Principally for paracrine and autocrine functions

Growth factors – polypeptides that function through stimulation of cellular proliferation (hyperplasia) or cell size
(hypertrophy)
Pathologies

Anorexia – fasting (low glucose levels) causes release of glucagon (normally released overnight) which mobilizes fuels from adipose tissue (glycogen stores) – process called glycogenolysis o Liver parenchymal cells have glucagon receptors, but skeletal muscle cells do not o Chronic fasting or exercise stimulates release of cortisol from adrenal cortex
 Cortisol synthesized and released in response to ACTH released due to chronic stress (pain, hypoglycemia, hemorrhage, exercise, etc.)
 Acts on tissues to change enzyme levels and redistribute nutrients in preparation for acute stress (increases transcription of genes for regulatory enzymes in gluconeogenesis o Exercise releases epinephrine and norepinephrine

Myasthenia gravis – autoimmune disease in which patient develops antibodies against nicotinic ACh receptors in skeletal muscle – results in muscle weakness o B and T lymphocytes cooperate in producing various antibodies against nicotinic ACh receptor o Treated with edrophonium chloride (inhibitor of acetylcholinesterase) so that ACh is allowed to accumulate in synaptic cleft (more ACh means more chances of binding to the few receptors left)

Diabetes – insulin increases general protein synthesis, which strongly affects muscle mass through hypertrophy o Insulin also regulates immediate nutrient availability and storage, including glucose transport into skeletal muscle and glycogen synthesis o Patients with type I diabetes mellitus who lack insulin rapidly develop hyperglycemia once insulin levels drop too low and also exhibit muscle wasting o Signal transduction pathway diverges after activation of receptor and phosphorylation of IRS, which has multiple binding sites for different signal-mediator proteins

Cholera – cholera A toxin absorbed into intestinal mucosal cells, where it is processed and complexed with ADPribosylation factor (Arf)

o Cholera A toxin is NAD-glycohydrolase that cleaves NAD and transfers ADP ribose portion to other proteins – as a consequence, G proteins remain actively bound to adenylyl cyclase, resulting in increased production of cAMP o Cystic fibrosis transmembrane conductance regulator (CFTR) channel activated, resulting in secretion of
Cl and Na + into intestinal lumen – ion secretion followed by loss of water, resulting in vomiting and diarrhea (causes dehydration)
Intracellular Transcription Factor Receptors

Small hydrophobic molecules diffuse through plasma membranes of cells to receptors for lipophilic messengers
(gene-specific transcription factors) bind specifically in DNA to regulate expression of certain genes

Gene-specific transcription factors include steroids, thyroid hormones, retinoic acid, and vitamin D – transported in blood bound to serum albumin or more specific transport proteins (steroid hormone binding globulin (SHBG) or thyroid hormone binding globulin (TBG)) o Intracellular receptors part of steroid hormone/thyroid hormone superfamily of receptors o Primarily localized to nucleus (some in cytoplasm) o Binding of hormone changes activity and ability to associate with or dissociate from DNA
Plasma Membrane Receptors and Signal Transduction

All plasma membranes receptors have o Extracellular domain that participates in binding of chemical messenger o One or more membrane-spanning domains that are α-helices o Intracellular domain that initiates signal transduction

Ligand binds to extracellular domain, causing conformational change that is communicated to intracellular domain through α-helix of transmembrane domain o Activated intracellular domain initiates characteristic signal transduction pathway that usually involves binding specific intracellular signal transduction proteins

Two major types of effects on cell o Rapid and immediate effects on cellular ion levels or activation/inhibition of enzymes o Slower changes in rate of gene expression for specific set of proteins

Major classes of plasma membrane receptors o Ion channel receptors (most small-molecule neurotransmitters and some neuropeptides use these – similar in structure to nicotinic ACh receptor) o Receptors that are kinases or that bind to and activate kinases
 Protein kinases transfer phosphate group from ATP to hydroxyl group of specific amino acid side chain in target protein – intracellular kinase domain of receptor activated when messenger binds to extracellular domain
 Receptor kinase phosphorylates amino acid residue on receptor or associated protein, and message is propagated downstream through signal transducer proteins that bind to activated messenger-receptor complex (STAT, Smad, or Src homology) o Heptahelical receptors (G protein-coupled receptors or GPCR) – contain 7 membrane-spanning α-helices and are most common type of plasma membrane receptor
 Work through second messengers such as cAMP generated inside cell in response to messenger binding to receptor
Signal Transduction through Tyrosine Kinase Receptors

Tyrosine kinase receptors – generally exist in membrane as monomers with single membrane-spanning helix o One molecule of growth factor generally binds 2 molecules of receptor and promotes dimerization o Once receptor dimer forms, intracellular tyrosine kinase domains of receptor phosphorylate each other on certain tyrosine residues, which form specific binding sites for signal transducer proteins

Ras-MAP kinase pathway – one of domains of receptor containing phosphotyrosine residue forms binding site for intracellular proteins with specific 3D structure (SH2 domain) o Adaptor protein Grb1 is one of proteins with SH2 domain that binds phosphotyrosine residues on growth factor receptors o Binding to receptor causes conformational change in Grb2 that activates SH3 domain that binds to SOS
(son of sevenless), which is a guanine nucleotide exchange factor (GEF) for Ras, a monomeric G protein located in the plasma membrane

 o SOS catalyzes exchange of GTP for GDP on Ras, causing conformational change in Ras that promotes binding of protein Raf (serine protein kinase) that begins phosphorylation cascade (when one of kinases in cascade is phosphorylated, it binds and phosphorylates next enzyme downstream) o MAP kinase cascade ultimately leads to alteration of gene transcription factor activity, thereby either upregulating or downregulating transcription of genes involved in cell survival and proliferation
Phosphatidylinositol signaling molecules can be generated through tyrosine kinase receptors or heptahelical receptors o Phosphatidylinositol phosphates functions
 Phosphatidylinositol 4’,5’-bisphosphate (PI-4,5-bisP) can be cleaved to generate 2 intracellular second messengers (DAG and IP
3
)
 PI-3,4,5-trisP can serve as PM docking site for signal transduction proteins that contain pleckstrin homology (PH) domain o PI 3-kinase contains SH2 domain and is activated by binding to specific phosphotyrosine site on tyrosine kinase receptor or receptor-associated protein o PTEN catalyzes dephosphorylation of PI-3,4,5-trisP to PI-4,5-bisP, removing primary signal from pathway
 Mutations in PTEN or misexpression of PTEN can lead to cancer

Insulin receptor – member of tyrosine kinase family of receptors – exists in membrane as preformed dimer, with each half containing α and β subunits o β-subunits autophosphorylate each other when insulin binds, activating receptor o activated receptor binds protein (insulin receptor substrate or IRS) o Activated receptor kinase phosphorylates IRS at multiple sites, creating multiple binding sites for different proteins with SH2 domains o Insulin receptor can also transmit signals through direct docking with other signal transduction intermediates
Signal Transduction by Cytokine Receptors: Use of JAK-STAT proteins

Tyrosine-kinase-associated receptors in cytokine receptor family transduce signals through JAK-STAT proteins to regulate proliferation of certain cells involved in immune response


Receptors bind tyrosine kinase of JAK family – signal transducer proteins (STATs) are of JAK family
Cytokine receptors utilize more direct route for propagation of signal to nucleus than tyrosine kinase receptors o As cytokine binds to receptors, they form dimers and may cluster o Activated receptor-associated tyrosine kinases phosphorylate each other on tyrosine residues and phosphorylate tyrosine residues on receptor, forming phosphotyrosine-binding sites for SH2 domain of
STAT (inactive in cytoplasm until they bind to receptor complex where they are phosphorylated by bound JAK) o Phosphorylation changes conformation of STAT, causing it to dissociate from receptor and dimerize with another phosphorylated STAT, forming an activated transcription factor o STAT dimer goes to nucleus and binds to response element on DNA, regulating gene transcription

Different cytokines target different genes because different STATs cause dimers of different kinds to form

Suppressors of cytokine signaling (SOCS) and protein inhibitors of activated STAT (PIAS) regulate STAT signaling
Receptor Serine-Threonine Kinases

Associate with proteins from Smad family, which are gene-specific transcription factors

Includes TGF-β and BMPs
Signal Transduction through Heptahelical Receptors

Have no intrinsic kinase activity but initiate signal transduction through heterotrimeric G proteins composed of
α, β, and γ subunits

Heterotrimeric G proteins – use hormone that activates adenylyl cyclase (e.g., glucagon, epinephrine) o When α-subunit contains GDP, it remains associated with β and γ-subunits, either free in membrane or bound to unoccupied receptor o When hormone binds, conformation change in receptor promotes GDP dissociation and GTP binding – exchange of GTP for bound GDP causes dissociation of α-subunit from receptor and from β and γsubunits o α and γ-subunits tethered to intracellular side of PM through lipid anchors, but can still move around on membrane surface

o GTP-α-subunit binds its target enzyme in membrane, changing its activity (binding and activating adenylyl cyclase, increasing synthesis of cAMP) o With time, Gα-subunit inactivates itself by hydrolyzing its own bound GTP to GDP and P i
(unrelated to number of cAMP molecules formed) o α-subunit containing GDP dissociates from target protein and reforms trimeric G protein complex, which is then free to bind an empty hormone receptor o because of “internal clock”, continued activation of receptor is necessary for continued signal transduction and elevation of cAMP o G proteins through which internal clock has become defective can become permanently activated, continuously stimulating signal transduction pathway

When hormone binds and adenylyl cyclase activated, it synthesizes cAMP from ATP o cAMP hydrolyzed to AMP by cAMP phosphodiesterase, which resides in PM o Concentration of secondary messengers kept at very low levels in cells by balancing activity of cyclase and phosphodiesterase so that cAMP levels can change rapidly when hormone levels change (insulin lowers cAMP levels by causing phosphodiesterase activation) o cAMP is allosteric activator of PKA (serine-threonine protein kinase that phosphorylates large number of enzymes, providing rapid response to hormones such as glucagon and epinephrine) o hormone cross-talk – interconnections between different kinase pathways

Heptahelical receptors bind q isoform of Gα-subunit, which activates target enzyme phospholipase C
β
, which hydrolyzes membrane lipid PI-4,5-bisP into DAG and 1,4,5-IP
3 o IP
3
has binding site in sarcoplasmic reticulum and ER that stimulates release of Ca 2+ that activates enzymes containing calcium-calmodulin subunit o DAG remains in membrane and activates PKC, which propagates response by phosphorylating target proteins
Changes in Response Signals

Intracellular phosphorylation sites alter receptors’ ability to transmit signals

Receptor number varied through downregulation o After hormone binds to receptor, hormone-receptor complex may be taken into cell by clathrindependent endocytosis o Receptors may be degraded or recycled back to surface under conditions of constant high hormone levels when more receptors are occupied by hormones
Signal Termination

Many chronic diseases caused by failure to terminate response at appropriate time

Transduction pathway can be terminated by chemical messenger itself – when stimulus is no longer applied to secreting cell, messenger is no longer secreted, and existing messenger is catabolized o Example: insulin is taken up into liver and degraded

Signal may be turned off at specific steps

Pathway for reversing message typically utilized by receptor kinase pathways is protein phosphatases (enzymes that reverse action of kinases by removing phosphate groups from proteins)