Transcript Signal Transduction - Clayton State University

Lecture 08 (Chapter 13)
Signal Transduction
Signal Transduction
• Signal-transducing receptors are frequently plasma membrane
proteins that bind specific extracellular molecules
• Growth factors
• Hormones
• Neurotransmitters
• Binding transmits signal to cell interior which initiates response
(e.g., enzyme cascades)
3 Major Classes of Signal-transducing receptors
• Ion channel linked receptors
Extracellular recognition domain,
• Protein linked receptors
transmembrane domain,
• Enzyme linked receptors
intracellular domain
Complete structures (with all domains) are not available for these receptors due to
problems with crystallization, and large size which prevents study by NMR
Signal Transduction
• Signal-transducing receptors are frequently plasma
membrane proteins that bind specific extracellular molecules
• Growth factors
• Hormones
• Neurotransmitters
http://pharmaceuticalintelligence.com/tag/signal-transduction-pathways/
G proteins
•
•
•
•
G proteins, (guanine nucleotide-binding proteins), are a family of protein receptors
that act as molecular switches inside cells.
Receive input signal from outside of the cell.
Signal is transmitted across membrane into the cytosol, and amplified.
These signal mediate several important physiological responses related to vision,
smell, and stress response.
(1) ligand activates the G protein-coupled
receptor. (2) Induces a conformational
change in the G-protein allowing it to
function as a guanine nucleotide
exchange factor (GEF) that exchanges GTP
for GDP (4) - thus turning the GPCR "on".
The GTP (or GDP) is bound to the Gα
subunit in the traditional view of
heterotrimeric GPCR activation. (5)This
exchange triggers the dissociation of the
Gα subunit (which is bound to GTP) from
the Gβγ dimer and the receptor as a
whole.
As long as ligand remains bound to GPCR,
production of Gα-GTP molecules will
continue (signal amplification).
http://en.wikipedia.org/wiki/G_protein
Structure of G Protein
•
•
•
Most G proteins are heterotrimers with Gα
(45 kDa), Gβ (35 kDa) and Gγ (8 kDa)
subunits.
G protein dissociates into the Gα subunit
(which is bound to GTP) and the Gβγ
subunits, which both transmit the signal to
different effector proteins.
The human genome contains code for over
1000 of these receptors, which can affect a
multitude of physiological processes.
Physiological processes mediated by G proteins
Stimulus
Receptor
Epinephrine
Beta-adrenergic receptor
Light
rhodopsin
IgE-antigen complexes mast cell IgE receptor
Effector
adenylate cyclase
c-GMP phosphodiesterase
phospholipase C
Acetylcholine
K channel
muscarinic receptor
http://swift.cmbi.ru.nl/gv/GPCR/rvdkant/GPCR/Introduction.html
Physiological response
glycogen breakdown
visual excitation
histamine secretion in all
allergic reactions
slowing of pacemaker
activity that controls
heartbeat
Structural basis of signal transduction by G Proteins
• Ras (abbreviation for Rat sarcoma) proteins
structurally similar to Gα subunit of G proteins
• Contains a six-stranded beta sheet and 5 alpha
helices
• G domain (166 amino acids) which binds
guanosine nucleotides.
• C terminal membrane targeting region (CAAXCOOH, also known as CAAX box)
• The G domain contains five G motifs that bind
GDP/GTP directly
•
•
•
•
•
•
G1 - P-loop binds the beta phosphate of GDP and GTP
G2 – (Switch I), T35 binds the terminal phosphate (γ-phosphate) of GTP and the divalent
Magnesium ion present in the active site, but makes no contacts with GDP
G3 – (Switch II), has the DXXGQ motif, where D57 is specific for guanine binding rather than
adenine and Glutamine-61 activates a catalytic water molecule for hydrolysis of GTP to GDP.
G4 - LVGNKxDL motif, provides specific interaction to guanine
G5 - SAK consensus sequence, A146 is specific for guanine rather than adenine
The switch motifs, G2 and G3 move upon activation by GTP. This conformation change mediates
the basic functionality as a molecular switch protein. This GTP bound state of Ras is On-state and
GDP bound state is the Off-state.
Introduction to Protein Structure, Branden & Tooze, 1999
Ras-regulated signal pathways
•
Ras-regulated signal pathways control
a number of processes, including:
– Regulation of signal transduction
processes leading to cell
multiplication and differentiation,
cell adhesion, apoptosis, and cell
migration
– Acting as molecular switches in
response to protein tyrosine
kinase receptors
• Activated by GTP binding, and
deactivated by GDP binding.
• About 25% of all tumor cells have mutant Ras proteins that cannot be activated to
hydrolyze GTP (deregulated)
• Remain bound to GTP and activated, thus leading to uncontrolled cell growth and
decreased apoptosis
http://www.genecopoeia.com/product/search/pathway/h_rasPathway.php
G protein transducin and rhodopsin
Rhodopsin
•
•
•
Light-sensitive receptor protein and biological pigment in photoreceptor cells of the
retinal rod cells (convey ability to see under limited light).
Consists of the protein moiety opsin and a covalently bound cofactor, retinal.
Light energy induces conformational change in retinal, which activates the G protein
transducin and subsequent cascade transforming light into neural signal via the optic
nerve.
http://cherfan2010biology12assessment.wikispaces.com/The+Retina
Rhodopsin
Opsin
Retinal
GDP
G tγ
Transducin
G tβ
G tα
Rhodopsin
• Rhodopsin consists of the protein
moiety opsin and a reversibly
covalently bound cofactor, retinal.
• Opsin is a bundle of seven
transmembrane helices connected
to each other by polypeptide loops.
• Opsin binds retinal (a photoreactive
chromophore), which is located in a
central pocket on the seventh helix
at a lysine residue.
• The retinal lies horizontally with
relation to the membrane. Each
outer segment disc contains
thousands of visual pigment
molecules.
Opsin
Retinal
http://en.wikipedia.org/wiki/Rhodopsin; http://de.wikipedia.org/wiki/Rhodopsin#/media/File:1L9H_(Bovine_Rhodopsin)_2.png
Retinal
•
•
•
•
•
•
•
•
http://en.wikipedia.org/wiki/Rhodopsin
Retinol is produced in the
retina from Vitamin A, from
dietary beta-carotene.
Retinol converted to retinal.
When lLight photon interacts
with retinal in photoreceptor
cell, retinal isomerizes from the
11-cis to all-trans configuration.
Retinal no longer fits into the
opsin binding site.
Opsin undergoes
conformational change to
metarhodopsin II.
Metarhodopsin II is unstable
and splits, yielding opsin and
all-trans retinal.
The opsin activates the
regulatory protein transducin.
11-cis-retinal is restored and
rhodopsin reassembled.
Visual Phototransduction via transducin
Light activation of opsin
http://webvision.med.utah.edu/book/part-ii-anatomy-and-physiology-of-the-retina/photoreceptors/
Visual Phototransduction via transducin
Transducin, activated by opsin, dissociates from its bound GDP, binds GTP.
Gtα dissociates from Gtβ and Gtγ subunits, with the GTP still bound to Gtα
http://webvision.med.utah.edu/book/part-ii-anatomy-and-physiology-of-the-retina/photoreceptors/
Visual Phototransduction via transducin
The Gtα-GTP complex activates phosphodiesterase (PDE).
PDE breaks down cGMP to 5'-GMP. This lowers the concentration of cGMP and
therefore the sodium channels close.
http://webvision.med.utah.edu/book/part-ii-anatomy-and-physiology-of-the-retina/photoreceptors/
Visual Phototransduction via transducin
Closure of the sodium channels causes hyperpolarization of the cell due to the
ongoing efflux of potassium ions.
Hyperpolarization of the cell causes voltage-gated calcium channels to close.
http://webvision.med.utah.edu/book/part-ii-anatomy-and-physiology-of-the-retina/photoreceptors/
Phototransduction Animation
http://sites.sinauer.com/neuroscience5e/animations11.02.html
Human Growth Hormone (HGH)
•
•
•
•
Peptide hormone that stimulates growth, cell reproduction and regeneration.
It is a mitosis-triggering protein which is specific only to certain kinds of cells.
Single chain polypeptide consisting of 191-amino acids.
Synthesized, stored, and secreted by somatotropic cells within the lateral wings of
the anterior pituitary gland.
HGH stimulates production of Insulinlike growth factor 1 (IGF-1), which
stimulates systemic body growth, and
has growth-promoting effects on
almost every cell in the body,
especially:
• skeletal muscle
• cartilage
• bone
• liver
• kidney
• nerves
• skin
• hematopoietic cell
• lungs
https://www.cgl.ucsf.edu/Outreach/midasplus/gallery/hGH.html
Human Growth Hormone Receptor
• Growth hormone receptor is a transmembrane receptor for growth hormone
protein.
The growth hormone receptor: mechanism of activation and clinical implications, Brooks J & Waters MJ, Nature Reviews Endocrinology, 2010
Human Growth Hormone Receptor and JAK2
•
•
•
Binding of asymmetrical growth hormone to the receptor leads to receptor dimerization and
the activation of an intra- and intercellular signal transduction pathway leading to growth.
The intracellular domain contains a proline-rich motif, termed ‘box 1’, that is required for the
binding and activation of janus kinase 2 (JAK2).
JAK2 has been identified as a tyrosine kinase associated with GHR and other receptors of the
superfamily. Binding of GH to its receptor results in dimerization of the GHR, phosphorylation
of JAK2 and of the GHR.
https://www.cgl.ucsf.edu/Outreach/midasplus/gallery/hGH.html, Growth hormone receptor:
structure and signal transduction, Postel-Vinay MC1, Finidori J., Eur J Endocrinol. 1995
GHR Pathway
• Phosphotyrosine residues on both GHR and JAK2 provide docking sites for a variety of
signalling proteins that contain phosphotyrosine-binding motifs (such as SH2 domains).
• The GH-bound receptor induces JAK2 phosphorylation of the various target proteins, leading
to different cellular responses.
http://www.ebi.ac.uk/interpro/potm/2004_4/Page2.htm
Enzyme-Linked Receptors
Classes
1. Receptor tyrosine kinases
–
Phosphorylate specific tyrosine residues on intracellular signaling
proteins
2. Tyrosine kinase-associated receptors
–
(e.g., GHR) associate with proteins that have tyrosine kinase activity
3. Receptor protein tyrosine phosphatases
–
Remove phosphate groups from tyrosine residues of intracellular
signaling proteins
4. Transmembrane receptor serine/threonine kinases
–
Add phosphate groups to serine or threonine
5. Transmembrane guanyl cyclases
–
Catalyze production of cyclic GMP in cytosol
Receptor tyrosine kinases (RTKs)
1.
2.
3.
4.
5.
6.
7.
Single-pass transmembrane receptors.
Intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the γphosphate of ATP to tyrosine residues in protein substrates.
Essential components of signal transduction pathways that affect cell
proliferation, differentiation, migration and metabolism.
Part of large family that includes:
–
insulin receptor
–
epidermal growth factor
–
fibroblast growth factor
–
vascular endothelial growth factor
Receptor activation occurs through ligand binding (growth factors, cytokines, and
hormones), which facilitates receptor dimerization and autophosphorylation of
specific tyrosine residues in the cytoplasmic portion.
The phosphotyrosine residues either enhance receptor catalytic activity or
provide docking sites for downstream signaling proteins.
May play critical roles in the development and progression of many types of
cancers.
Structural analysis of receptor tyrosine kinases, Stevan R Hubbard, Progress in Biophysics and Molecular Biology, 1999
http://schlessinger.yalemedicine.org/picture1.htm
Insulin Receptor (IR or InsR)
1.
Transmembrane (RTK) receptor activated by hormones
–
insulin
–
Insulin-like growth factor 1 (IGF-I)
–
Insulin-like growth factor 2 (IGF-II)
2. Regulates glucose homeostasis
–
Loss of function can lead to development of diabetes and
cancer.
3. Transcription of alternative splice variants derived from the INSR
gene are translated to form one of two monomeric isomers; IR-A
in which exon 11 is excluded, and IR-B in which exon 11 (12
additional Aas) is included.
4. Downstream post-translational modification to IR-A or IR-B result
in the formation of a proteolytically cleaved α and β subunits,
which can be combined to form homo or hetero-dimers leading to
production of the disulfide-linked transmembrane insulin
receptor.
Ward CW, Lawrence MC (April 2009). "Ligand-induced activation of the insulin receptor: a multi-step process involving structural changes in both
the ligand and the receptor". BioEssays; Belfiore A, Frasca F (Oct 2009). "Insulin receptor isoforms and insulin receptor/insulin-like growth factor
receptor hybrids in physiology and disease.". Endocr Rev
Insulin
• Peptide hormone produced by beta cells in the
pancreas.
• Regulates the metabolism of carbohydrates
and fats by promoting the absorption of
glucose from the blood to skeletal muscles and
fat tissue and by causing fat to be stored rather
than used for energy.
• Inhibits production of glucose by the liver.
• Except in certain metabolic disease states (e.g.,
diabetes mellitus and metabolic syndrome),
insulin is provided within the body in a
constant proportion to remove excess glucose
from the blood.
• Active insulin is a monomer.
• Inactive insulin binds zinc to form a hexamer.
http://en.wikipedia.org/wiki/Insulin; Sonksen P, Sonksen J (2000), "Insulin: understanding its
action in health and disease". Br J Anaesth
Each isometric monomer is organized
into 8 distinct domains:
• leu-rich repeat domain (L1),
• cys-rich region (CR),
• leu-rich repeat domain (L2),
• Three fibronectin type III
domains; Fn0, Fn1 and Fn2.
• insert domain (Ins) is found in
Fn1, containing the α/β furin
cleavage site, from which
proteolysis results in both IDα
and IDβ domains.
• Within the β-chain, lies the Fn2
domain, transmembrane helix
(TM) and intracellular
juxtamembrane (JM) region, just
upstream of the intracellular
tyrosine kinase (TK) catalytic
domain, responsible for
subsequent intracellular signaling
pathways.
• Structure stabilized by disulfide
bonds
Insulin Receptor Domain Structure
Structural biology of insulin and IGF1 receptors: implications for drug design, Pierre De Meyts & Jonathan Whittaker, Nature Reviews Drug
Discovery 1, (October 2002)
http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-14/CB14.html
Receptor protein tyrosine phosphatases - RPTPs
• RPTPs are enzymes that regulate a variety of cellular
processes including:
– cell growth
– differentiation
– mitotic cycle
– oncogenic transformation
• Specific ligands that bind RPTPs remain to be identified.
• Once activated, the receptor phosphatases remove phosphate
groups from phosphorylated tyrosine residues on proteins.
http://www.ncbi.nlm.nih.gov/gene/5786 ;
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753797/
Protein tyrosine phosphatases: from genes, to function, to disease, Nicholas K. Tonks, Nature Reviews Molecular Cell Biology 7, 833-846
(November 2006)
RPTPα
•
•
RPTPα contains an extracellular domain, a single transmembrane segment and
two tandem intracytoplasmic catalytic domains (D1 and D2).
RPTPα dephosphorylates and activates Src family non-receptor tyrosine kinases,
and is implicated in the regulation of integrin signaling, cell adhesion and
proliferation.
Receptor subtype R4
Proximal
PTP domain D1
Distal
PTP domain D2
1yfo
http://www.ncbi.nlm.nih.gov/gene/5786 ; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753797/; Protein tyrosine phosphatases: from
genes, to function, to disease, Nicholas K. Tonks, Nature Reviews Molecular Cell Biology 7, 833-846 (November 2006)
RPTPα
Phosphorylation of PTPα by PKCδ causes detachment of Grb2 followed by
attachment of pp60src, effectively through SH2 domain exchange.
Binding of the SH2 domain of pp60src to PTPα exposes phosphotyrosine 527 in
pp60src (yellow star), which is then dephosphorylated by PTPα.
Protein tyrosine phosphatases: from genes, to function, to disease, Nicholas K. Tonks, Nature Reviews Molecular Cell Biology 7, 833-846
(November 2006)