Membrane Function Signal Transduction

I. Introduction to Receptors & Signal Transduction

The Players l Signaling molecules l Receptors l G-proteins l Second messenger systems l Effector proteins

Signaling Molecules l Neurotransmitters l Hormones l Growth factors l Drugs l Other nomenclature  Ligand  Agonist / Antagonist

Receptors l Receptors are proteins associated with cell membranes l Receptors “recognize” signaling molecules by binding to them. l Binding of receptors by signaling molecules ---> Cell behavior change

Figure 1: Overview of Signaling Tyrosine Kinase mRNA Synthesis Protein Synthesis Second Messangers Protein Kinases Ion Channels Hormones: Steroids Thyroid Growth Factors Transmitters Hormones

Neurotansmitters: Biogenic Amines. l Catecholamines  Epinephrine  Norepinephrine  Dopamine l Esters:Acetylcholine l Indolamines  Histamine  5-HT

Neurotransmitters: Peptides l Substance P l Neuropeptide Y (NPY) l Enkephalins l Somatostatin l VIP

Neurotransmitters: Amino Acids l Excitatory  Glutamate  Aspartate l Inhibitory   -aminobutyric acid (GABA)  Glycine

Neurotranmitters: Other l Nitric Oxide l Arachadonic acid l Carbon Monoxide l PAF l Zinc

The G-Proteins l Involved in most signaling processes l Link receptor proteins to effector proteins. Trimeric proteins composed of , , and  -subunits.

Figure 2: G-Protein Cycling Adenylate Cyclase Phospholipase C Ion Channels Phospholipase A 2 Phosphodiesterase             A A A A R R R R GTP (GTPase) -P i GTP GDP

Functional G-Protein Units GTP-activated  -subunit  produce second messenger  and/or opens ion channels.  -complexes  Initially thought to be inert.  Probably not inert  Exact role currently ill-defined.

Second messengers produced by G-protein activation. l Adenylate Cyclase  cAMP l Phospholipase C (PLC)  Inositol triphosphate (IP 3 )  Diacylglycerol (DAG) l Ion Channel Activity

Families of G-proteins Unique structure of their  -subunits.  subunits appear to be similar across families. l Main families:  G  s  G  i  G  q

II. cAMP Second Messenger System

Figure 3: Adenylate Cyclase Adenylate Cyclase R1R1 R2R2 AsAs GsGs GiGi AiAi GTP GDP GTP GDP PDE AMP cAMP ATP-Mg ++ Reg C C C C Protein Protein-P Protein Kinase A (PKA) PKA

Summary of Adenylate Cyclase Activation l Receptors which associate with G s -type G-protein  Stimulates adenylate cyclase.  Increases cAMP l Receptors which associate with G i -type G-protein  Inhibit adenylate cyclase.  Decreases cAMP

Summary of cAMP action l cAMP exerts its effect by activating protein kinase A (PKA) l PKA phosphorylates proteins  Enzymes, pumps, and channels  Phosphorylation can either increase or decrease activity depending on the protein.

Adenylate Cyclase l Family of membrane spanning enzymes. l Types I through IV have been well characterized.  Additional types probably exist. l Types differ with respect to activity modulation by other second messenger systems

Adenylate Cyclase Activity and Other Messenger Systems l Kinases (PKA, PKC, other) can phosphorylate adenylate cyclase in some cells. l Binding of adenylate cyclase by:   -subunits of other G-proteins  Ca ++ /calmodulin complexes l Allows other second messenger systems to interact with cAMP system

III. The Phospholipase C Second Messenger System: IP 3 and DAG

Figure 4: Phospholipase C System R Ca ++ PKC Ca ++ Endoplasmic Reticulum GqGq PLC Protein Protein-P A DAG IP 3 PIP 2

Summary of the Phospholipase C Messengers l Agonist binds receptor l Occupied Receptor ---> activation of PLC (G q -mediated) l PLC Produces second messengers: IP 3 and DAG l PLC activation associated with Ca ++ -channel activation

Action of IP 3 l IP 3 binds to IP 3 -receptors on the endoplasmic reticulum l Releases intracellular Ca ++ stores.

Action of DAG l Remains membrane associated. l Activates Protein kinase C (PKC) which translocates from the cytosol to the membrane l Activated PKC phosphorylates other proteins and alters their function state.

PLC System and Calcium l PLC causes the IP 3 -mediated Calcium l PLC also causes the influx of Ca ++. l Ca ++ binds one of a family of Ca ++ binding proteins (calmodulin). l Ca ++ /calmodulin complex  binds to yet other proteins and changes their functional activity.

IV. Guanylate Cyclase: cGMP and Nitric Oxide As Second Messengers

Figure 5: Nitric Oxide and cGMP cGMP NO Ca ++ GTP GMP Intracellular Ca ++ Stores Ca ++ Arginine + Citrulline GTP NO PDE Membrane Bound Guanylate Cyclase Soluble Guanylate Cyclase C.M. Ion Channels cGMP-Dependent PK PDEase Activity NO Synthetase

NO is Membrane Soluble. l Diffusion to nearby cells l Increase cGMP levels in nearby cells l Vascular endothelial cells and nearby smooth muscle cells.

V. SIGNALING BY ACETYLCHOLINE

Acetylcholine As a Neurotransmitter Acetylcholine As a Neurotransmitter l Both the central and peripheral nervous systems. l Binds two broad classes of receptors:  Nicotinic receptors  Muscarinic receptors.

Nicotinic Receptor Features l Composed of 5 subunits:  2 , ,  and . l Subunits are arranged to form a central cavity that extends across the membrane. l Nicotinic receptors are also channels l ACh-binding opens gates and allows ion fluxes across the channel

Figure 6: Nicotinic Receptor Channel Agonist Binding Site Gate

Subclasses of Nicotinic Receptors l Skeletal muscle (N 1 or N m )  Unique  and  subunits l Autonomic ganglia (N 2 or N g ). l Both N 1 and N 2 are gene-product families not single receptor types.

Other Ligand-Gated Channels l Structural and sequence similarity to nicotinic receptors. l Example agonists for these channels include:  Serotonin (5-HT)  Glutamate  GABA  Glycine

Muscarinic receptors l Muscarinic receptors are not channels. l Operate through G-proteins to alter second messenger systems. l 5 muscarinic subtypes have been cloned and sequenced (M 1, M 2, M 3, M 4, M 5 ).

Grouping Muscarinic Receptors l M 1, M 3, and M 5 receptors: Activate Phospholipase C through Gq.  PLC activation ---> increased IP 3 --> increased intracellular Ca ++  Increased intracellular Ca++ ---> Activation of Ca ++ -sensitive K + & Cl - channels.

Grouping Muscarinic Receptors l M 2 and M 4 receptors  G i -coupled inhibition of adenylate cyclase  G o or G i -coupled regulation of certain Ca ++ & K + channels.

VI. Signaling by Epinephrine and Norepinephrine and Coupling Through Adrenergic Receptors

Three Families of Adrenergic Receptors:  -receptors: Three subtypes      and  .   -receptors: Three subtypes   A    B  and   C   -receptors: Three subtypes   A     B  and   C

. All adrenergic receptors appear to be coupled to cellular processes through G-proteins

Occupation of  Adrenergic Receptors l G s -mediated stimulation of adenylate cyclase l Increased cAMP l Increased PKA activity.

Occupation of   -Adrenergic Receptors l Mechanistic details sketchy l Possibly G q -mediated PLC activation  Increases IP 3 and DAG for some subtypes (1B)?  Activates Ca ++ -channels for other subtypes (1A)?

Occupation of   -Adrenergic Receptors l G i -mediated inhibition of adenylate cyclase. l Decreased cAMP l Decreased PKA activity.