Endocrinology 2 CELL SIGNALLING helen.christian

Cell Signalling Week 1 HT2012
Endocrinology 2
Chapter 2 ‘How drugs act’ Rang & Dale Pharmacology
Chapter 15 ‘Cell communication’ Alberts Molecular Biology of the Cell
Weatherman et al 2006 ‘Untangling the estrogen receptor web’ Nature Chem Biol 2:175
Lania et al 2006 ‘Mechanisms of disease: mutations of GPCR’ Nature Clin Practice 2:681
These articles and others of interest can be found on Weblearn ‘Endocrinology’ pages in Organisation of Body
Core learning objectives
Define a receptor and its basic properties
Describe the form and location of the principal types of receptors
Describe the main different mechanisms of action used by drugs/hormones, and how these influence membrane
properties, enzyme activity in the cell, gene transcription
How do hormones act?
Hormones influence specific target cell activity. Most hormones are present in the circulation at very low
concentrations i.e. 10-12 - 10-9M. The target cells express specific, high affinity receptors that recognise and
bind the hormone and mediate the cellular effect. A single hormone can have many actions on/in one target
cell type. There may be different subtypes of receptor for a hormone in different or the same tissues.
Principal types of hormone receptor
Plasma membrane receptors are integral (glyco)proteins, that is the receptors span the lipid bilayer and can
thus signal to the interior of the target cell. Receptors are freely mobile within the membrane.
Three types of surface receptors:
Ion channel linked – e.g. nicotinic ACh receptor
Open ion channels and change electrical activity of target cell.
G-protein linked – many examples, including adrenergic receptors, glucagons receptors.
Conformational change in receptor is transmitted to the G-protein on the cytoplasmic side of the
membrane, and G protein in turn regulates the activity of membrane bound enzyme(s) which
generate second messenger molecules within the cell.
Enzyme linked –
Tyrosine kinase-linked e.g. insulin, growth hormone receptors. Ligand binding stimulates
autophosphorylation of a domain contained in the receptor structure, which activates the receptors
enzyme activity and allows it to phosphorylate tyrosine residues on target proteins .
Guanylate cyclase-linked e.g. atrial natriuretic factor, guanylate cyclase produces cyclic GMP which
activates kinases.
Insulin receptor
Adrenaline, glucagon receptor
tyrosine kinase
Activates protein kinase cascade to phosphorylate
target proteins, causing cell function to be altered
Cell Signalling Week 1 HT2012
Intracellular receptors linked to gene transcription. The receptors are in the cytoplasm or nucleus.
Control gene expression and in turn protein levels to activate many cellular effects. e.g. receptors for
steroid hormones, thyroid hormones, vitamin D derivatives.
G-Protein Coupled Receptors (GPCR’s)
Structure: 7-transmembrane domain proteins.
Activating Systems:
Receptors are coupled via G proteins (guanosine triphosphate (GTP)-binding proteins) to enzymes that
produce >second messengers’.
The interaction of receptor with hormone leads to a conformational change in the receptor.
The G protein is then able to interact with intracellular portions of the receptor and in turn activates
enzymes that produce second messengers.
G-proteins have a trimeric structure made up of , , and  subunits.
 The  subunits bind GTP. Different classes of G-protein exist:
 Gs - activate adenylate cyclase to increase formation of cAMP.
 Gi - inhibit adenylate cyclase (decrease cAMP).
 Gq - activate phospholipase C.
The G protein cycle
G proteins interconvert between an inactive GDP form and an active GTP-bound form. The exchange of
GTP for bound GDP is catalysed by an active receptor with hormone bound. G-GTP activates the effector
proteins (e.g. adenylate cyclase or PLC). Hydrolysis of the bound GTP brings the G protein back into the
inactive state. The whole cycle is driven by the energy of GTP hydrolysis.
Second messengers e.g. cAMP, cGMP, IP3 & DAG, raised Ca2+ are:
- derived from abundant precursors (ATP, GTP, membrane phospholipids, Ca2+ stores).
- rapidly generated by hormone action .
- formed in small amounts, but produce amplification of initial hormone signal.
- rapidly removed (phosphodiesterase; inositol lipid metabolism; Ca2+ pumps).
Adenylate cyclase: produces cyclic AMP (cAMP) from ATP.
 Gs stimulates (e.g. -adrenergic, LH receptors) and Gi inhibits (e.g. somatostatin receptors)
adenylate cyclase activity.
 cAMP activates protein kinase A (PKA).
 PKA is a tetramer, consisting of two regulatory subunits (R) and two catalytic subunits (C). In the
absence of cAMP, R2C2 is inactive. The binding of cAMP to the R chains releases the C chains
from the R2 complex, which are then catalytically active.
 Activated PKA phosphorylates specific serine and threonine residues in many target proteins to alter
their function.
 Examples of target proteins include:
 enzymes controlling glycogen formation/breakdown.
 proteins involved in exocytosis of hormones.
 gene expression via transcriptional activator cAMP-response element binding protein (CREB).
Phospholipase C: produces inositol triphosphate (IP3) and diacylglycerol (DAG) from PIP2.
 the role of the G protein Gq is to stimulate phospholipase C.
 IP3 - mobilises intracellular calcium; affects Ca2+ channels.
 DAG - activates protein kinase C.
 The fatty acid chain arachidonate of PIP2 is the precursor of the prostaglandins, so activation of the
Cell Signalling Week 1 HT2012
IP3/DAG pathway may give rise to many more molecules with signalling roles.
What does an increase in Ca2+ from resting levels (~0.1M to 1M) do in the cell?
Direct activation of Ca2+ dependent processes e.g. protein kinases, exocytosis
Binds to calmodulin, a Ca2+ binding protein . Ca2+-calmodulin stimulates a wide range of enzymes,
pumps and other target proteins, including:
 calmodulin-dependent protein kinases (CaM kinase II) – phosphorylates many different
proteins, regulating fuel metabolism, ionic permeability, neurotransmitter release
 Ca2+-ATPase pump is also activated by Ca2+-calmodulin binding, restoring calcium to basal
level in the cell
Summary of the actions of G-protein linked receptors
protein kinase
dependent protein kinases
Binds to
Other Ca2+-calmodulin
dependent processes
Examples of hormones acting via cAMP
Adrenaline (2), glucagon, vasopressin (V2),
thyroid-stimulating hormone
luteinising hormone
follicle stimulating hormone
Release of
Ca2+ stores
kinase C
Other Ca2+-dependent
via IP3/DAG
adrenaline (, vasopressin (V1)
angiotensin II, GnRH
Cholera toxin covalently modifies the Gs subunit such that it cannot hydrolyse GTP, and thus becomes
permanently activated. This causes a continuous intestinal secretion, leading to diarrhoea and dehydration,
which rapidly become life-threatening.
Pertussis toxin (from the organism that causes whooping cough) inactivates Gi unit of G-proteins by
stabilising the GDP-form, so removing the inhibitory effect on adenylate cyclase.
The effectors stimulated by G-protein activation provide an amplification system. The binding of one
hormone molecule to one receptor activates many G-proteins. Each of these in turn can act on many
effector molecules, each of which can produce many second messenger molecules. The second messengers
can activate many protein kinases. These can phosphorylate and thus modulate the activity of many target
How are the signalling pathways switched off?
1. Removal of signal – if hormone level in blood drops.
2. Internalisation of receptor-ligand complex into cell for recycling - receptors are uncoupled in
endosomes; lysosomes break down some receptors, some recycling occurs via the Golgi.
3. Desensitisation of receptors.
Cell Signalling Week 1 HT2012
4. Breakdown of second messengers – IP3 and DAG are relatively short lived molecules; cAMP is
broken down by phosphodiesterase.
5. Reversal of the modification of the target e.g. dephosphorylation of a kinase target.
Tyrosine kinase-linked receptors
Single transmembrane proteins with intrinsic or linked kinase activity (e.g. growth
factor/GH/insulin/cytokine receptors).
Intracellular domain contains tyrosine kinase activity that is activated following ligand binding and
receptor dimerisation.
Tyrosine kinase phosphorylates proteins, especially transcription factors e.g. JAK and STAT cascades
for mediating Growth Hormone effects.
Intracellular hormone receptors – regulate gene transcription e.g. steroid/thyroid/vit D receptors
The receptor contains DNA-binding region (zinc fingers) and hormone-binding regions.
The receptors are localised in the nucleus or cytoplasm.
Hormone binding induces receptor activation and migration to DNA transcription sites.
The activated receptor interacts with hormone-response elements (HREs) in the promotor, the
regulatory part of the target gene.
 The receptor-ligand complexes mostly form dimers.
(Steroid hormones also have rapid actions via putative membrane receptors)
Steroid receptor structure and mechanism of action
Mutations may:
 cause the receptor to be absent or abnormal and therefore inactive - hormone resistance conditions
alter receptor affinity/stability, receptor-effect coupling (e.g. inactivating mutations of the GHRH
receptor cause pituitary dwarfism).
 make receptors active in the absence of the hormone ligand (e.g. activating mutations of the LH
receptor cause precocious puberty).
 anti-receptor antibodies can either inactivate the receptor, or switch it on (e.g. Grave’s).
Hormone resistance can arise in disease by downstream defects in hormone action (e.g. insulin resistance
in adult onset diabetes).
Syllabus: 14.1.1 The mechanisms of action of hormones; Regulation of enzyme activity; 3.1.4
regulation of gene expression; 4.2 Response (applies equally to hormones as to drugs).
Any questions email: helen.christian@dpag.ox.ac.uk