Download Welcome to Biochemistry/Endocrinology

Mechanism of hormone
action, signal transduction, 2nd
messengers and receptors
Reading material Devlin 6e Chapter 13
MAHMOUD A. ALFAQIH BDS PhD
Department of Physiology & Biochemistry
Jordan University of Science & Technology,
School of Medicine
Overview of Hormones
• Mobile signals secreted by endocrine
system for distant cell-cell
communication
• Occur as four major classes (and several
minor ones)
– Peptide hormones: tiny “proteins” from 3200 aa’s, water-soluble, active only after
cleavage of the targeting “pre-sequence”
and the inactive “prohormone” (insulin,
glucagon)
Continue………….
Overview of Hormones
– Amino acid derivative hormones: derived
from amino acids, water-soluble, some are
neurotransmitters (epinephrine, T3, T4 )
– Eicosanoids: derived from arachidonate,
minimally water-soluble, act locally (rather
than through the bloodstream), mediate
pain, inflammation
– Steroid hormones: derived from
cholesterol, fat-soluble, are carried to
targets by carrier proteins
Common Characteristics of Hormones
• They occur and function at very low
concentrations – 10-6 to 10-12 M
• Deliberately unstable – levels rise rapidly
upon secretion, but fall fast when it stops
• Biochemical response may be very rapid, by
altering existing enzyme activities, or slower,
where gene expression levels change
• Act through two receptor types: cell surface
and nuclear
• Display remarkable specificity
• Operate through the “cascade” principle
6
Two Types of Hormone Receptor
• Hormones are present at very low
concentration, thus receptor
affinity must be very tight
• Cell surface receptors bind
hormone outside – a
conformational change
transduces the signal to the
inside, resulting in a “second
messenger” (e.g. cAMP)
• Cytosolic/nuclear receptors bind
and retain hormone, migrating to
the nucleus as a hormonereceptor complex and then alter
gene expression
Figure 13.6 Structures of four second -messenger molecules.
Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc .
An Overview of Hormone Classes
1.
2.
10
Receptor tyrosine kinase
Receptor serine/threonine kinase
Figure 13.5 Basic characteristics of receptor subtypes as functionally distinct receptor proteins that bind a
common extracellular signaling molecule.
Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc .
Mechanism of hormone action, signal transduction, 2nd
messengers and receptors
ENZYME LINKED RECEPTORS
12
04.12.8
Enzyme linked receptors;
Receptor tyrosine kinases (RTKs)
 Include receptor for insulin, Epidermal Growth
factor (EGF), platelet derived growth factor
(PDGF)
 Most are single subunit receptors but some
exist as multimeric complexes (Like insulin)
 Amino end is extracellular (Ligand binding),
carboxyl end is intracellular and contains
catalytic domain
Enzyme linked receptors;
Receptor tyrosine kinases (RTKs)
 Ligand binding induces receptor dimerization and activates
catalytic domain
 Catalytic domain auto-phosphorylates receptor (receptor also
functions as an effector protein)
 Phosphorylated tyrosine residues become anchoring point for
adaptor proteins
 Adaptor proteins have special protein-protein interaction
domains called (SH2 for SRC Homology 2)
 RTKs allow accumulation of many types of tyrosine
phosphorylated proteins (secondary effector proteins)
Insulin, EGF, PDGF
Figure 13.14 Conformational and functional changes in a receptor tyrosine kinase during activation by growth
factor binding.
Redrawn based on figure from Alberts, B., et al., Molecular Biology of the Cell, 4th ed. New York: Garland Science, 2002.
Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc .
Mutations in RTKs can lead to
cancer
Figure 13.15 Mutated forms of receptor tyrosine kinases as the products of cancer-causing oncogenes.
Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc .
GTP binding regulatory proteins
 They could be functionally coupled to “G protein
coupled receptor” (Exist as trimers)
 They could exist as functionally distinct monomeric
proteins
 They have GTPase activity which allows them to
function as “molecular switches”
 Exchange of GDP with GTP activates the proteins
17
GTP binding regulatory
proteins
18
Ras GTPase and MAP Kinases
 Ras is a small monomeric GTP binding regulatory protein
 About 30% of all tumors have mutations in Ras GTPase
 Adaptor proteins can directly bind to phosphorylated RTKs and
recruit Ras activating proteins
 Ras activating proteins enhance GDP exchange with GTP
 Ras protein can then activate downstream signaling pathways
involved in proliferation (MAP kinase cascade)
Figure 13.16 Role of the ras GTPase during intracellular signal transduction by an activated receptor tyrosine
kinase.
Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc .
Ras GTPase and MAPK
Figure 13.17 Role of the MAP kinase cascade during intracellular signal transduction by an activated receptor
tyrosine kinase.
Redrawn based on figure from Alberts, B., et al., Essential Cell Biology, 2nd ed. New York: Garland Science, 2004.
Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc .
Signaling by protein
phosphorylation
22
Receptor Serine/Threonine Kinase
 Receptors for transforming growth factor B
(TGFB)
 Important role in cell signaling during tissue
differentiation and fetal development
 Mutations in TGFB mediated signaling
pathway leads to cancer
23
Receptor Serine/Threonine
kinases
Figure 13.18
Redrawn based on figure from Alberts, B., et al., Essential Cell Biology, 2nd ed. New York: Garland Science, 2004.
Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc .
Mechanism of hormone action, signal transduction, 2nd messengers
and receptors
G PROTEIN COUPLED
RECEPTORS
25
G protein coupled receptors
(GPCRs)
 GPCRs bind to a diverse range of ligands (proteins, peptides,
amino acid derivatives, lipids, nucleotides)
 Play an important role in endocrine, paracrine, autocrine
signaling in all tissues and cell types
 Sensory proteins just like rhodopsin are GPCRs
 GPCRs, their ligands and their downstream pathways are targets
for many of the currently used drugs
 GPCRs have a great degree of structural but not sequence
similarity
26
GPCRs
27
04.12.8
Heterotrimeric G proteins
 GPCRs are functionally coupled to trimeric complexes
consisting of α, β and γ.
 α subunit is the largest subunit, hydrophilic and
contains GTPase activity
 Twenty subtypes of α subunit are grouped into
subfamilies based on specific downstream action
 αs Subunit stimulates adenylate cyclase, while αi
inhibits it
28
Heterotrimeric G proteins
Figure 13.21 Major subclasses of heterotrimeric G proteins that are activated by G by the seventransmembrane domain G-protein-couple receptors.
Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc .
Heterotrimeric G protein cycle
30
Termination of signaling by GPCRs
31
Cyclic AMP based signal
transduction
 The use of cAMP as a second messenger is largely
limited to GPCRs
 Cells regulate both the synthesis and the degradation
of cAMP
 cAMP synthesis is catalyzed by adenylate cyclase
effector enzyme
 An integral membrane protein
 At least six genes that encode different subtypes have
been identified
Cyclic AMP based signal
transduction
 All subtypes are activated by αs Subunit of G proteins
and also inhibited by αi subunit
 Breakdown of cAMP is catalyzed by a variety of
phosphodiesterases that hydrolyze ester bond and
lead to the production of AMP
 Activity of phosphodiesterase is hormonally regulated
and is affected by drugs
 Caffeine inhibits activity of phosphodiesterases
leading to accumulation of cAMP
Breakdown of cAMP
34
Adenylate cyclase activity
Protein kinase A
• Activated adenylyl cyclase makes cAMP, the “2nd
messenger”,
• Which activates “PKA” which phosphorylates other proteins,
Such as phosphorylase kinase, which in turn
Phosphorylates glycogen phosphorylase
• This mobilize glucose from liver glycogen!
Intracellular signaling mechanism
of cAMP
36
Turn off of the signal:
1. Ga hydrolyzes GTP to GDP + Pi.
(GTPase).
The presence of GDP on Ga causes it to
rebind to the inhibitory bg complex.
Adenylate Cyclase is no longer activated.
2. Phosphodiesterase catalyzes
hydrolysis of
cAMP  AMP.
Turn off of the signal (cont.):
3. Receptor desensitization occurs. This process
varies with the hormone.
 Some receptors are phosphorylated via specific
receptor kinases.
 The phosphorylated receptor may then bind to a
protein arrestin, that promotes removal of the
receptor from the membrane by clathrin-mediated
endocytosis.
4. Protein Phosphatase catalyzes removal by
hydrolysis of phosphates that were attached to
proteins via Protein Kinase A.
Signal amplification is an important
feature of signal cascades:
One hormone molecule can lead
to formation of many cAMP
molecules.
Each catalytic subunit of Protein
Kinase A catalyzes
phosphorylation of many proteins
during the life-time of the cAMP.
Activation of PKA
heterotetramer
(inactive)
dimer
active monomers
Cyclic GMP based signal transduction
 Levels of cGMP are regulated by a balance between
guanylate cyclase and cGMP phosphodiesterase
 Guanylate cyclase could be either membrane bound
or a free cytosolic protein (soluble)
 Membrane bound guanylate cyclase could be
activated by ANF (atrial natriuretic factor)
 Soluble Guanylate cyclase is activated by NO (nitric
oxide)
41
Cyclic GMP based signal transduction
 Activation of guanylate cyclase leads to relaxation of
vascular smooth cells through activation of cGMP
sensitive protein kinases
 Mechanism:
1. Attenuation of calcium release from sarcoplasmic
reticulum
2. Activation of myosin light chain phosphatases
3. Activation of potassium channels leading to
hyperpolarization of myocyte membrane
42
Signaling mechanism of cGMP
43