Download Introduction to Hormone Signalling Receptors and signals theme

Introduction to Hormone
Signalling
Receptors and signals theme
‘Hormone action’
Signalling in Health and Disease 2011
GPCR
Tues Wk1
Enzyme - linked receptors G protein - coupled receptors
Drug discovery
Thurs Wk2
Receptors and
drugs
Tues Wk 2
nuclear receptors
Insulin and Growth
factor signalling
Hilary term
Peptide hormone G protein linked receptors
Light
amines
Ions
Amino acids
Peptides
Lipids
Enzymes
Odorants
Hypothalamic
hormones:
GnRH
Somatostatin
CRH
TRH
GHRH
AVP
Oxytocin
Pituitary
hormones:
ACTH
LH
FSH
TSH
MSH
Gut hormones:
Secretin
CCK
Glucagon
Parathyroid hormone
Substance P
Opiates
NPY
Bombesin
Neurotensin
Angiotensin II
PACAP/VIP
Glucagon-like peptide 1
Calcitonin
hCG
Receptor molecular
biology/bioinformatics
Thurs/Fri Wk1
Second
messengers
Mon Wk 2
Genomic and nongenomic steroid
signalling
Hilary term
GnRH and LH receptor structure
GnRH receptor no Cter tail
3D
LH receptor long Nter tail
Antisense knockout of Gα
αo prevents TRH
stimulation of Ca2+ currents in a pituitary cell line
G protein cycle
Which G protein?
Drugs – cholera
toxin
Find mRNA of G protein
TRH
Prepare complementary
oligonucleotide probe
TSH
Inject probe into cell
Antisense knockout
of specific subunits
in cell lines
Probe binds to mRNA preventing
protein translation
Protein ‘knocked out’
Examine functional activity of cell
(electrical activity, hormone release)
Laugwitz et al, 1996 PNAS
tissue
expression
pattern
bioinformatics
Evidence for
GPCR
dimerisation
Fluorescence
Resonance
Energy
Transfer
Salaphour 2000
full length
cloning
Reverse
pharmacology
approach to
characterizing
orphan GPCR
expression
putative
ligands;
biological
extracts
functional
screens,
e.g. cAMP
natural or
surrogate
agonist
antagonist
pharmacology of
receptor
Transcription
via cAMP
Clinical evidence of a role for GPCRs in
normal and aberrant growth
Activating mutations
TSH
thyroid adenoma, hyperthyroidism
LH
pseudoprecocious puberty
GHRH
acromegaly
Inactivating mutations
CREB = cAMP
response
element binding
protein
Greenspan & Strewler
GHRH signalling is essential for post-natal
expansion of the GH cell population
VP V2
Diabetes insipidus
TSH
Congenital hypothyroidism
LH
Leydig cell hypoplasia
GHRH
Pituitary dwarfism
Inactivating Mutation in the Growth Hormone-Releasing
Hormone Receptor in man: Dwarfism of Sindh
MAHESHWARI et al., (J Clin Endocrinol Metab 1998 83: 4065–4074)
GH cells
No
response to
GHRH
normal
GHRH receptor
mutation
GH cells
lit/lit
mouse
Lin et al, Nature
364, 208 (1993)
Grave’s disease
Enzyme-linked receptors
Predominantly signal growth and
differentiation
tyrosine kinase linked e.g. insulin, EGF
cytokines, growth factors e.g. GH, PRL
Growth factor receptors e.g. insulin
tyrosine
kinase
activation
Autophosphorylation
of the receptor
SH2
GRB2 SOS
GRB2 binds via SH2
domain and
provides
‘scaffolding’ for
signal apparatus
MAPK
MAPK
SRF
AP1
RAS RAF1
MEK1
Transcription
Insulin Signalling activates different
pathways in parallel
Convergence of GPCR and
tyrosine kinase linked receptor
pathways
Cytokine receptors e.g. GH, PRL
GH
GH
JAK2
JAK2
tyr
tyr
JAK2
JAK2
tyr
P
P
tyr
Ligand activated receptor
recruits cytosolic tyr kinase
phosphorylation of
downstream effectors
e.g. STAT, MAPK
‘Classical’ mechanism of
steroid action
Steroid receptor structure
Estrogen receptor
Onset >30min
Steroid receptor
plasma
membrane
N ter
AF-1
NLS
C ter
A/B
C
DNA
control of gene
transcription
HRE X
gene X
steroid
nucleus
mRNA
HRE = hormone response element
protein
FUNCTION
AF-2
D
E
F
ligand
agonists
A/B
protein-protein interactions, transcriptional activation ‘transactivation’
C
zinc fingers important for DNA binding
D
Nuclear localisation signal
E
Ligand binding, heat shock protein binding, transcription repression,
receptor dimerisation
F
Present in few receptors, minimal function
Steroid receptor stabilises the preinitiation complex for transcription
Steroid mediated induction of
protein synthesis
Chick oviduct
IIJ
IIF
SRE SRE
IIA
IIB
IIE
IIH
progesterone
DES
mRNA
oviduct weight
total protein
TATA
ovalbumin
pol II
general
transcription
factors
IIJ
IIF
SRE SRE
IIA
IIB
IIE
IIH
lysozyme
avidin
mRNA
TATA
pol II
O’Malley, 1995
Steroid receptors interact with
co-regulator proteins
ubiquitin ligase, kinase, acetylase
coactivation
help to recruit pol II
O’Malley, 1969
Partial hormone
resistance in
SRC1 KO males
WT
Compensation for lack
of SRC-1 by increasing
expression of other
coactivators
enzyme
remodel chromatin
Science (1995)
Xu et al, 1998
KO
Positive and negative factors on
steroid receptor pathway
Role of orphan steroid receptors
ERR = estrogen receptor related receptor
Level
Enhancers
Opposers
receptor
ligand
ligand independent
activators
HSPs
ligand binding
protiens
phosphorylation
kinases
phosphatases
heterodimerization
RXRs
COUP-TF etc
nuclear translocation ATP chaperones
ATP depletion
transactivation
coactivators
corepression
chromatin structure
histone acetylase
histone deacetylase
KO demonstrates a role in early placentation
Luo et al, 1997
Rapid action of estradiol
in the anterior pituitary
24
**
**
*
8
**
**
**
16
12
**
10 min
E
E-BSA
4
[K+ ]
Basal
0
-12 -11 -10 -9 -8
log [Steroid] (M)
-7
Christian & Morris, J Physiol 2002
-6
Mean FL1 intensity (units)
Ir
Ir--PRL (ng mg-1 tissue)
20
120
100
80
60
control
+ 1 µM E
40
20
0
1
10
100
BSABSA- 0.1
FITC [E[E-BSABSA-FITC] (nM)
100 nM
What are the steroid-binding sites in the
plasma membrane?
Palmitoylation of steroid receptors
important for membrane localisation
Receptors derived from the nuclear R?
Evidence for:
effects not evident in receptor knockout tissue
immunocytochemistry using antibodies
effects blocked by nuclear R antagonists
Novel ‘orphan’ receptors? GPCR30- estrogen
Modulation of known existing receptors?
GABA – neurosteroids, integrin –thyroid hormones
2007
Mutation of residues that are palmitoylated reduces membrane localisation
ER associated with cytosolic face of membrane not transmembrane
Novel G protein
coupled receptor
GPR30 binds
estrogen
Early membrane estrogenic effects
required for full expression of slower
genomic effects in a nerve cell line
Neuroblastoma cell
line
See Weatherman et al
Nature Chem Biol 2:175
(on Weblearn)
Transfected with
luciferase reporter
gene with promotor
containing estrogen
response elements
Measure reporter
gene activity
Pulse 1 20 min with estrogen
Rest 4h
Pulse 2h with estrogen
Vasudevan et al 2001
Evidence that membrane effects of
estrogen permit optimal nuclear
effects
‘Although non-classical and genomic
mechanisms have been widely viewed as
discrete, alternate models of steroid hormone
action these data show that they can synergize
to potentiate transcription’
Concn response curve
Vasudevan et al 2001
Conclusion
• Hormones have multiple effects with
actions in the membrane complementing
those in the nucleus
• Actions at the cell membrane are not
exclusively mediated by peptide
hormones
Vasudevan et al 2001