L20 - Signalling Via Intracellular Receptors Flashcards
Why are some signalling molecules intracellular?
Some signal molecules are hydrophobic or small enough to pass across the plasma membrane and interact directly with intracellular enzymes or receptors
What are two examples of signalling via intracellular receptors?
Nitric oxide
Lipid soluble signalling molecules e.g. retinoic acid
Receptors bound by lipid soluble signalling molecules are?
Structurally related and form the nuclear receptor superfamily
What are the two types of receptors involved in signalling via intracellular receptors?
Intracellular receptors
Nuclear receptors
What are intracellular receptors?
Nitric oxide (NO•) Carbon monoxide (CO) Often have carrier proteins associated with them
What are nuclear receptors?
Steroid hormones
Thyroid hormones
Retinoids
Vitamin D
What are the 3 key properties of nitric oxide?
Gaseous
Free radical - unpaired electron in outer shell
Very reactive - half-life 5-10s
What are the 3 side molecules produced in the two successive oxidations to produce nitric oxide?
L-arginine –> N-hydroxyarginine –> L-citrulline + NO
What enzyme catalyses the oxidations to produce nitric oxide?
Nitric oxide synthase (NOS)
What sort of expression does constitutive NOS have?
Expression constitutive – expressed all the time
What is constitutive NOS induced by?
Enzymatic activity induced by Ca2+/calmodulin
When Ca activates them the activity is short-lived
- Produces picomoles of NO
When activated what does constitutive NOS form?
Picomoles of NO
What are the two types of constitutive NOS?
eNOS – endothelial
nNOS – neural
Where is eNOS expressed?
Bound at cell membrane Endothelium Cardiac myocytes Renal mesangial cells Osteoblasts/osteoclasts Platelets
Where is nNOS expressed?
Found in the cytoplasm CNS NANC neurons ENS Retina
What is induced NOS induced by?
Transcriptionally induced in response to pathological stimuli - LPS, IFN-g, IL-1
What kind of activity does inducible NOS have?
Enzymatic activity constitutive – active all the time
What kind of expression does inducible NOS have?
Expression inducible
When induced they have a longer lasting effect
Produce nanomoles of nitric oxide
When activated what does inducible NOS form?
Nanomoles of nitric oxide
Where is inducible NOS expressed?
Macrophages and Kupffer cells
Neutrophils
Fibroblasts
Vascular smooth muscle and endothelial cells
What is the effect of nitric oxide in the vasculature?
- Autonomic nerves release Ach - acts on eNOS
- eNOS release nitric oxide - causes surrounding smooth muscle to relax - vasodilation
- NO inhibits platelet aggregation and vascular smooth muscle proliferation
a. Anti-atherosclerotic
How is nitric oxide induced?
- Acetylcholine activates nitric oxide synthase (cNOS) in endothelial cells
- NOS generates nitric oxide from arginine
- Nitric oxide activates the production of cGMP
What does nitric oxide activate the production of?
cGMP
How does nitric acid activate the production of cGMP?
- cGMP
- cGMP – dependent protein kinase
- Myosin light chain phosphatase
- Myosin light chain
What is the effect of nitric oxide in the brain?
- In the CNS, nNOS is tethered close to NMDA-type glutamate receptors
- This means it can respond to Ca2+ increases near the open channels
a. Implications for synaptic plasticity - Glutamate is produced by a presynaptic terminal and this activates glutamate receptors
- Especially NMDA receptors which stimulate the nitric oxide pathway
- The nitric oxide produced travels to the presynaptic terminal
- It increases the production of glutamate - creating the state of long-term potentiation
What is the role of nitric oxide in the immune system?
Kills bacteria and parasites
Induces programmed cell death
Triggers inflammatory diseases when overproduced in lungs and intestines
- Rheumatoid arthritis, Crohn’s disease and asthma
Role in tumour apoptosis and necrosis
What does iNOS in inflammatory cells use nitric oxide for?
Use nitric oxide as a cytostatic and cytotoxic agent
Involves much higher amounts of NO
How was nitroglycerine use as a pharmaceutical?
100 yr treatment for angina - pain due to inadequate blood flow to heart
Nitroglycerine breaks down in vivo to generate nitric oxide which relaxes blood vessels
- Decreases load on heart
- Increases heart blood supply
What is dynamite made of?
Nitroglycerine + fine clay as a stabiliser
What does nitric oxide activate in smooth muscle?
Guanylyl cyclase which leads to formation of cGMP
How can too much cGMP in a cell be fixed?
Phospho-diesterase 5
Converts cGMP to GMP sing cGMP dependent protein kinase
How does Viagra work?
- Inhibits phosphodiesterase 5
- Leads to less breakdown of cGMP
- Smooth muscles constricting blood vessels relax
- More blood flow
What are some examples of the nuclear receptor superfamily?
Cortisol Estradiol Testosterone Thyroxine Vitamins D3 Retinoic acid
How many domains do nuclear receptors have?
3 Transcription activating domain DNA binding domain Regulatory binding domain - Binds to ligands releasing inhibitory protein
How do nuclear receptors go from their active state to inactive state?
- Inhibitory proteins hold the receptors in inactive state
- Binding to ligand removes inhibitors leading to a conformational change
- Receptors now interact with coactivators to direct transcription
The effects of the ligands are modified in different tissues by the expression of different coactivators
What is the structural organisation of the estrogen receptor?
DNA binding domain consists of Zinc Fingers
Each finger binds a specific DNA sequence
Examples of symmetric homodimers
Glucocorticoid and estrogen receptors
Examples of heterodimers
Vitamin D, thyroid hormone, and retinoic acid receptors
How do symmetric homodimers bind?
Bind as symmetric homodimers to an inverted repeat DNA site
How do heterodimers bind?
Bind as heterodimers to direct repeat sites
RAR:RXR or VDR:RXR
Where are homodimeric receptors found?
Anchored in the cytoplasm by inhibitor proteins in the absence of ligand
- E.g. Hsp90
Hormone binding causes release of inhibitor proteins allowing entry to nucleus
Where are heterodimaeric receptors found?
Located exclusively in nucleus
How do heterodimeric receptors act in the absence of ligands?
Act as repressors in the absence of ligand - recruitment of histone deacetylases
How do heterodimeric receptors act in the presence of ligands?
Act as activators in the presence of ligand - recruitment of histone acetylases
Early primary response to steroid hormone – 1-6 hours
Steroid-hormone receptor complexes activate transcription of primary response genes
Some of these genes will encode transcription factors
Induced synthesis of a few proteins
Delayed secondary response to steroid hormones – 6-48 hours
Primary response protein shuts of primary response genes and turn on secondary response genes
Induced synthesis of secondary response proteins
