Physio Lecture 5 – Intracellular signaling Prof. dr. Željko Dujić.

Physio Lecture 5 – Physio Lecture 5 – Intracellular signalingIntracellular signaling

Prof. dr. Željko Dujić

INTRACELLULAR SIGNAL TRANSDUCTION:A Journey from the Plasma Membrane to the Nucleus

(with interesting stops along the way)

INTRACELLULAR SIGNALING:Signal Transduction

Cell membranes, as well as the cell cytoplasm and even the cell nucleus, contain cell-specific receptors for various ligands, which are involved in outside-inside signaling, i.e. signal transduction.

Ligands include hormones, growth factors, cytokines, prostaglandins and proteases.

Hormones are involved in a variety of metabolic processes that maintain homeostasise.g. fuel metabolism. Particularly noteworthy in that regard are glucagon, insulin and the catecholamines (epinephrine and norepinephrine).

Growth factors are involved in mitogenesis, whereas cytokines play critical roles in thedifferentiation, proliferation and function of various cell lineages..

Interaction of such ligands with their membrane, cell-specific receptors or intracellularreceptors causes conformational changes in the receptor and, in many instances receptor-associated cytoplasmic proteins.

Such events result in the initiation of a cascade of important, but as yet incompletely understood, events leading to e.g. enzyme activation, differentiation and/or cell division.

Extracellular signalingmolecules released by cells occurs over distancesfrom a few microns - autocrine (c)and paracrine (b) signaling toseveral meters in endocrine (a)signaling. In some instances,receptor proteins attached to themembrane of one cell interactdirectly with receptors on anadjacent cell (d).

© 2000 by W. H. Freeman and Company. All rights reserved.

General Schemes of Intercellular Signalling

FORCES DRIVING SELECTION OF CURRENT MECHANISMS

Need for coordinated intercellular communication

Need to translate extracellular signals into series of intracellular events, while allowing for specificity

Specificity Determinants: 1. Specific receptors on or in the target cells recognize an appropriate ligand. 2. Specific response to receptor occupancy - effector pathways

Diversity of intercellular communication is achieved with hundreds of signaling molecules, including...

Proteins, small peptides, amino acids, nucleotides, steroids, fatty acid derivatives, and even dissolved gases such as NO and CO

Intracellular receptors:- signaling molecules include steroid hormones, retinoids, thyroxine, etc- receptor-hormone complex acts a transcription factor to alter transcription of certain genes

Cell surface receptors:- signaling molecules include peptide hormones, catecholamines, insulin, growth factors, cytokines, etc-binding, and subsequent events, triggers an or in the cytosolic concentration of a second messenger; or the activated,bound receptor acts as a scaffold to recruit and activate other intracellular proteins


Receptor Classes

ADVANTAGES1. Each cell is programmed to respond to specific combinations of signaling molecules.2. Different cells can respond differently to the same chemical signal.

Molecular Biology of the Cell, 2002

HORMONES - First class of signaling molecules defined

Secreted from endocrine cells - specialized signaling cells that control the behavior of an organism as a whole:

1. Differ from other intracellular mediators2. Usually stimulate metabolic activities in tissues remote from the secretory organ3. Active at very low concentrations (pM - M)4. Response to hormonal signal comes as a direct and rapid result of its secretion5. Metabolized rapidly so effects are, in most instances, short-lived, leading to rapid adaptations to metabolic changes

1. Peptides or polypeptides - insulin, glucagon, growth hormone, insulin-like growth factors, vasopressin, prolactin….2. Glycoproteins - follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH)…3. Steroids - glucocorticoids (aldosterone, cortisol), steroids (progesterone, testosterone), retinoic acid…4. Amino acid derivatives - epinephrine, norepinephrine, thyroxine, triidothyronine

Categories:

AGONISTS vs. ANTAGONISTS

Agonist mimics a hormone in binding productively to a receptor

Antagonist mimics a hormone stereochemically, but binds to the receptornon-productively, inhibiting the action of the natural hormone

Hormonebinds 2 receptor in lung

bronchial relaxation

Agoniste.g. important therapy

in asthma

Antagonistcontrol heart beat

binds 2 receptor in heart muscle increased heart rate

THREE LARGEST CLASSES OF CELL SURFACE RECEPTORS

GlucagonEpinephrineThrombin

InsulinGrowth factors


Intracellular Signaling Mediated by G protein-linked Membrane Receptorse.g. Glucagon, Epinephrine and Thrombin as signaling molecules

1. Activates a chain of events alterations in concentrations of signaling molecules; elaborate sets of interacting molecules that can relay signals from cell surface to the nucleus

2. Components: Receptor; Transducer (G protein): Effector (membrane-bound enzyme); Second messenger (e.g. cAMP); Response (cascade of highly-regulated protein phosphorylations, etc)

RECEPTOR: typically a seven transmembrane domain, integral membrane protein

Ligand binding domain(extracellular site)

-adrenergic receptor

Gs binding site(intracellular site)

All G protein-coupled receptorsresemble the -adrenergic receptorin their amino acid sequence and membranetopography.

Signal Transduction Pathway Activation of Adenylate Cyclase by Gs

Receptor: 7 TM domain integral membrane protein

Transducer: Gs

Effector: adenylate cyclase

Second messenger: cAMP

Cycle of G protein dissociation/association:

Activation of G proteins involves GTP displacement of GDP bound to the subunitand dissociation of the complex from the subunits, an exchange facilitated by a GEF protein(guaninenucleotide exchange factor) specific for Gs.

G protein activation

The active subunit binds to and activates/stimulates membrane-associated adenylate cyclasewhich catalyzes the conversion of ATP to cyclic AMP (cAMP).

Adenylate cyclaseactivation

The GTPase activity associated with the subunitslowly hydrolyzes the bound GTP to GDP which is facilitated by a GAP (GTPase activating protein) specific for Gs.

GTP hydrolysisThe subunit with GDP bound reassociateswith the subunits, and membrane associatedadenylate cyclase returns to its basal activity level

1. G protein is an trimeric protein which binds guanine nucleotides.2. They function to couple integral membrane receptors to target membrane-bound enzymes.3. They can be considered molecular switches wherein… GDP (inactive) GTP (active) +

4. The dissociated subunit expresses GTPase activity.

Characteristics of G proteins

5. GTPS blocks GTPase activity of GTP.

i withGDP bound

Region that changesconformation whenGTP is hydrolyzed


Receptor: 7 TM domain integral membrane protein

Transducer: Gs

Effector: adenylate cyclase

Second messenger: cAMP

Response: PKA regulatedprotein phosphorylations

Activation of cAMP-dependent protein kinase (PKA)cAMP binds to the PKA regulatory subunits conformational changes, which causes their dissociation from the catalytic subunits kinase activation. Release of the catalytic subunits requires the binding of more than two cyclic AMP molecules greatly sharpening the response of the kinase to changes in [cAMP].

PKA is a Ser/Thr kinase with discrete substrate specificity, thus facilitating a cascade of highly regulated protein phosphorylations.



Down stream effects:

1. Phosphorylation ofregulatory enzymes ofmetabolic pathways.

2. Increase transcriptionof certain proteins via phosphorylation of CREBleading to protein syn-thesis of target proteins.

How Increased Intracellular cAMP Leads to Increased Gene Transcription.

Activated PKA enters the nucleus and phosphorylatesCREB (cAMP ResponseElement Binding protein).Once phosphorylated, CREBrecruits the coactivator CBP (CREB Binding Protein). This complex binds to the CREB-binding element to stimulate gene transcription.



Down stream off signals:

1. cAMP hydrolyzedby phosphodiesterase

2. Ser/Thr phosphatasesdephosphorylate the phosphorylated targetproteins.

**

Early off signals:

1. Gs-associated GTPaseactivity GTP hydrolysisto GDP leading to re-association of GsGDPand dissociation of the hormone/receptor complex

Desensitization or Endocytosis of GPCR’s Effected by Phosphorylation

1. The ligand activated receptor can be phosphorylated on select Ser/Thr residues by GRK (e.g. BARK - adrenergic receptor kinase). These phosphorylated residues provide a docking site for arrestin resulting in inactivation/desensitization.2. In some instances, arrestin binding targets the receptor for clathrin-dependent endocytosis.3. In addition, if the occupied GPCR leads to cAMP, the receptor can also be phosphorylated by PKA leading to its inactivation/densensitization.

Clathrin-dependent Receptor-mediated endocytosis

Gs vs. Gi

Regulation of Adenylate Cyclase Activity

Gs stimulates adenylate cyclase

Gi inhibits adenylate cyclase

e.g. epinephrine can increase or decrease intracellular cAMP concentrations,depending upon the receptor to which it binds

adrenergic receptors couple to Gs, whereas

2 adrenergic receptors couple to Gi

Figure 21.38 – Devlin, Textbook of Biochemistry

Vasopressin binding to its GPCR activates Gs cAMP and activation of PKA.PKA phosphorylates various proteins the ultimate aggregation of microtubular subunits, which insert as water channels in the luminal plasma membrane to increase the reabsorption of water by free diffusion.

Images of cAMP Transients in Cultured Aplysia Sensory Neurons.

The cell was loaded with a fluorophore that would allow for the quantification of cAMP concentrations within the cell.

A: Free cAMP in the resting cell is < 5 X 10-8 M.B: Stimulation with serotonin, activates adenylate cyclase increasing cytoplasmic cAMP to ~ 1 X 10-6 M (red), especially within fine processes with a high surface to volume ratio. Thurs, within 20 sec of stimulation, the intracellular [cAMP] increased ~ 20-fold.

Inhibition of Gs and Gi by Bacterial Toxins

Cholera toxin effects on Gs:ADP ribosylation of an Arg residuein the s subunit of Gs inhibition ofassociated GTPase activity

Pertussis toxin effects on Gi:ADP ribosylation of a Cys residuein the i subunit of Gi an inabilityto inhibit adenylate cyclase activity.

Thus, both toxins cause increasedintracellular cAMP concentrations!


Gs vs Gi vs Gq

Gs and Gi coupled to adenylate cyclase [cAMP] G q coupled to phospholipase C [Ca2+]

Cellular mechanisms that maintain very low intracellular Ca2+ concentrationsA: Ca2+ is actively pumped out of the cytosol.

B: Ca2+ is pumped into the ER and mitochondria.

Cells must work very hard to maintain low intracellular [Ca2+] in order for Ca2+ to result in effective intracellular signaling.

INTRACELLULAR Ca2+ AS A “SECOND” MESSENGER

**


Ligand Binding to a GPCR Linked to Gq Phospholipase C Activation

Phospholipase C (PLC)-catalyzed hydrolysis of PIP2 Formation of 2 second messengers

**

**

Phospholipase C (PLC)-catalyzed hydrolysis of PIP2 Formation of 2 second messengers

IP3 release of Ca2+ from intracellular stores by binding to an IP3-gated Ca2+ channel in the endoplasmic reticulum.

DAG with released Ca2+ and membrane-associated phosphatidylserine activates Protein Kinase C (PKC - a Ser/Thr kinase). PKC directly phosphorylates intracellular proteins some of which gene transcription.

Ca2+ “Second” Messenger SystemRelease from intracellular stores subsequent to PLC-catalyzed hydrolysis of PIP2

1. Must be distinguished from cAMP-induced effects where cAMP activates a variety of Ca2+ channels Ca2+ influx from extracellular milieu (e.g. -adrenergic receptor occupancy in muscle cells Ca2+ influx rate and force of heart beat).

2. Increased intracellular [Ca2+] - Third messenger: Immediate vs. Sustained responses - Ca2+ binds to its ubiquitous intracellular receptor, calmodulin, thereby a) activating Ca2+/calmodulin-dependent kinases (CaM kinases), Ser/Thr kinases b) that have their own sets of substrate proteins, some of which when phosphorylated can or gene transcription.

Increased Intracellular Ca2+: A Third Messenger

Calmodulin in a ubiquitous intracellular Ca2+ receptorCa2+/calmodulin (CaM) complexes activate CaM-dependent kinases

(e.g. CaM-kinase II)


Structure of Ca2+/calmodulin complex based onX-ray diffraction and NMR spectroscopy studies


Protein Kinase C Activation of Gene TranscriptionTwo Independent Pathways


Down-regulation of Phosphoinositide-activated Second Messenger System

Off-signals:1. IP3 rapidly dephosphorylated by phosphatases.2. DAG rapidly hydrolyzed.3. Ca2+ rapidly pumped out.4. Ser/Thr phosphatases dephosphorylate PKC and CaM kinase targets.

Physio Lecture 5 – Intracellular signaling Prof. dr. Željko Dujić.

Documents

Transcript of Physio Lecture 5 – Intracellular signaling Prof. dr. Željko Dujić.