Intracellular Signal Transduction
Intracellular Signal Transduction
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 homeostasis e.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 the differentiation, proliferation and function of various cell lineages.. Interaction of such ligands with their membrane, cell-specific receptors or intracellular receptors causes conformational changes in the receptor and, in many instances receptorassociated 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 signaling molecules released by cells occurs over distances from a few microns - autocrine (c) and paracrine (b) signaling to several meters in endocrine (a) signaling. In some instances, receptor proteins attached to the membrane of one cell interact directly with receptors on an adjacent cell (d).
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
Receptor Classes
ADVANTAGES
1. Each cell is programmed to respond to specific combinations of signaling molecules. 2. Different cells can respond differently to the same chemical signal.
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 mediators 2. Usually stimulate metabolic activities in tissues remote from the secretory organ 3. Active at very low concentrations (pM - M) 4. Response to hormonal signal comes as a direct and rapid result of its secretion 5. Metabolized rapidly so effects are, in most instances, short-lived, leading to rapid adaptations to metabolic changes
Categories:
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
Antagonist
mimics a hormone stereochemically, but binds to the receptor non-productively, inhibiting the action of the natural hormone
Agonist
e.g. important therapy in asthma
binds 2 receptor in lung bronchial relaxation binds 2 receptor in heart muscle increased heart rate
Hormone
Antagonist
control heart beat
RECEPTOR CHARACTERISTICS
1. Participates in transduction of the signal from the external messenger to some component of the metabolic machinery 2. Has at least one additional functional site which is altered by ligand binding (allosteric site) 3. Ligand binding to receptors is saturable, resembling Michaelis-Menten kinetics
How to calculate a Kd and number of receptors from direct binding data Derivation of a Scatchard Plot
-adrenergic receptor
All G protein-coupled receptors resemble the -adrenergic receptor in their amino acid sequence and membrane topography. Gs binding site (intracellular site)
G protein activation
The subunit with GDP bound reassociates with the subunits, and membrane associated adenylate cyclase returns to its basal activity level
GTP hydrolysis
Characteristics of G proteins
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.
Down stream effects: 1. Phosphorylation of regulatory enzymes of metabolic pathways. 2. Increase transcription of certain proteins via phosphorylation of CREB leading to protein synthesis of target proteins.
Activated PKA enters the nucleus and phosphorylates CREB (cAMP Response Element Binding protein). Once phosphorylated, CREB recruits the coactivator CBP (CREB Binding Protein). This complex binds to the CREB-binding element to stimulate gene transcription.
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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.
Gs vs. Gi
Regulation of Adenylate Cyclase Activity
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. Figure 21.38 Devlin, Textbook of Biochemistry
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.
Gs vs Gi vs Gq
Gs and Gi coupled to adenylate cyclase [cAMP]
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Molecular Biology of the Cell, 2002
Cellular mechanisms that maintain very low intracellular Ca2+ concentrations A: Ca2+ is actively pumped out of the cytosol. B: Ca2+ is pumped into the ER and mitochondria.
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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.
Structure of Ca2+/calmodulin complex based on X-ray diffraction and NMR spectroscopy studies
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.