1. Chapter 34 The Reception and Transmission of Extracellular Information
to accompany
Biochemistry, 2/e by
Reginald Garrett and Charles Grisham
All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, Sea Harbor Drive, Orlando, Florida

2. Outline 34.1 Hormones and Signal Transduction Pathways
34.2 Signal-Transducing Receptors Transmit the Hormonal Message
34.3 Intracellular Second Messengers
34.4 GTP-Binding Proteins” The Hormonal Missing Link
34.5 The 7-TMS receptor

3. Outline 34.6 Specific Phospholipases
Calcium as a Second Messenger
Protein Kinase C
The Single TMS-receptor
34.10 Protein Modules
34.11 Steroid Hormones
SPECIAL FOCUS: Neurotransmission and Sensory Systems

4. (There may be others, but we doubt it...)
Classes of Hormones
(There may be others, but we doubt it...)
Steroid Hormones - derived from cholesterol- regulate metabolism, salt/water balances, inflammation, sexual function
Amino Acid Derived Hormones - epinephrine, etc.- regulate smooth muscle , blood pressure, cardiac rate, lipolysis, glycogenolysis
Peptide Hormones - regulate many processes in all tissues - including release of other hormones

5. Signal-Transducing Receptors Transmit Hormone Message
Non-steroid hormones bind to plasma membrane and activate a signal-transduction pathway inside the cell
Steroid hormones may either
bind to the plasma membrane or
enter the cell and travel to the nucleus

7. Types of Receptors Three that we know of...
7-transmembrane segment receptors
extracellular site for hormone (ligand)
intracellular site for GTP-binding protein
Single-transmembrane segment receptors
intracellular catalytic domain - either a tyrosine kinase or guanylyl cyclase
Oligomeric ion channels

8. Many and there may be more!
Second Messengers
Many and there may be more!
The hormone is the "first messenger"
The second messenger - Ca2+, cAMP or other - is released when the hormone binds to its (extracellular) receptor
The second messenger then activates (or inhibits) processes in the cytoplasm or nucleus
Degradation and/or clearance of the second messenger is also (obviously) important

9. cAMP and Glycogen Phosphorylase
Earl Sutherland discovers the first second messenger
In the early 1960s, Earl Sutherland showed that the stimulation of glycogen phosphorylase by epinephrine involved cyclic adenosine-3',5'-monophosphate
He called cAMP a "second messenger"
cAMP is synthesized by adenylyl cyclase and degraded by phosphodiesterase

12. How are the hormone receptor and AC coupled?
Purified AC and purified receptor, when recombined, are not coupled.
Rodbell showed that GTP is required for hormonal activation of AC
In 1977, Elliott Ross and Alfred Gilman at Univ. of Virginia discovered a GTP-binding protein which restored hormone stimulation to AC
Hormone stimulates receptor, which activates GTP-binding protein, which activates AC

14. Many new developments in this area
G Proteins
Many new developments in this area
Two kinds: "heterotrimeric G proteins" and "small G proteins"
X-ray diffraction structures for several of these are only recently available
Structures shed new light on possible functions

15. Heterotrimeric G Proteins
A model for their activity
Binding of hormone, etc., to receptor protein in the membrane triggers dissociation of GDP and binding of GTP to -subunit of G protein
G-GTP complex dissociates from G and migrates to effector sites, activating or inhibiting
But it is now clear that G also functions as a signalling device

17. Signalling Roles for G()
A partial list
Potassium channel proteins
Phospholipase A2
Yeast mating protein kinase Ste20
Adenylyl cyclase
Phospholipase C
Calcium channels
Receptor kinases

18. Stimulatory and Inhibitory G
G proteins may either stimulate or inhibit an effector.
In the case of adenylyl cyclase, the stimulatory G protein is known as Gs and the inhibitory G protein is known as Gi
Gi may act either by the Gi subunit binding to AC or by the Gi complex complexing all the Gi and preventing it from binding to AC
Read about the actions of cholera toxin and pertussis toxin

21. An oncogene and its product
The ras Gene and p21ras
An oncogene and its product
a gene first found in rat sarcoma virus
Normal cellular ras protein activates cellular processes when GTP is bound and is inactive when GTP has been hydrolyzed to GDP
Mutant (oncogenic) forms of ras have severely impaired GTPase activity, so remain active for long periods, stimulating
excessive growth and metabolic activity - causing tumors to form

24. Receptors that interact with G proteins
7-TMS Receptors
Receptors that interact with G proteins
Seven putative alpha-helical transmembrane segments
Extracellular domain interacts with hormone
Intracellular domain interacts with G proteins
Adrenergic receptors are typical
Note desensitization by phosphorylation, either by ARK or by protein kinase A

26. Phospholipases Release Second Messengers
Inositol phospholipids yield IP3 and DAG
PLC is activated by 7-TMS receptors and G proteins
PLC is activated by receptor tyrosine kinases (via phosphorylation)
Note PI metabolic pathways and the role of lithium

27. Other Lipids as Messengers
Recent findings - lots more to come
More recently than for PI, other phospholipids have been found to produce second messengers!
PC can produce C20s, DAG and/or PA
Sphingomyelin and glycosphingolipids also produce signals
Ceramide (from SM) is a trigger of apoptosis - programmed cell death

33. Ca2+ as a Second Messenger
Several sources of Ca2+ in cells!
[Ca2+] in cells is normally very low: < 1M
Calcium can enter cell from outside or from ER and calciosomes
CICR - Calcium-Induced Calcium Release - is very, very similar to what happens at the foot structure in muscle cells!
IP3 (made by action of phospholipase C) is the trigger
See Figures

37. M. Berridge's model of Ca2+ signals
Calcium Oscillations!
M. Berridge's model of Ca2+ signals
Ca2+ was once thought to merely rise in cells to signal and drop when the signal was over
Berridge's work demonstrates that Ca2+ levels oscillate in cells!
The purpose may be to protect cell components that are sensitive to high calcium, or perhaps to create waves of Ca2+ in the cell

39. Mediators of Ca2+effects in cells
Ca2+-Binding Proteins
Mediators of Ca2+effects in cells
Many cellular proteins modulate Ca2+ effects
3 main types: protein kinase Cs, Ca2+-modulated proteins and annexins
Kretsinger characterized the structure of parvalbumin, prototype of Ca2+-modulated proteins
"EF hand" proteins bind BAA helices

42. Transduction of two second messenger signals
PKC is activated by DAG and Ca2+
Most PKC isozymes have several domains, including ATP-binding domain, substrate-binding domain, Ca-binding domain and a phorbol ester-binding domain
Phorbol esters are apparent analogues of DAG
Cellular phosphatases dephosphorylate target proteins
Read about okadaic acid

47. Single TMS Receptors Three main classes
Extracellular domain to interact with hormone
Single transmembrane segment
Intracellular domain with enzyme activity
Activity is usually tyrosine kinase or guanylyl cyclase
Each of these has a "nonreceptor" counterpart
src gene kinase - pp60v-src was first known
Two posttranslational modifications

50. Receptor Tyrosine Kinases
Membrane-associated allosteric enzymes
How do single-TMS receptors transmit the signal from outside to inside??
Oligomeric association is the key!
Extracellular ligand binding

52. The Polypeptide Hormones
Common features of synthesis
All secreted polypeptide hormones are synthesized with a signal sequence (which directs them to secretory granules, then out)
Usually synthesized as inactive preprohormones ("pre-pro" implies at least two precessing steps)
Proteolytic processing produces the prohormone and the hormone

53. Proteolytic Processing
A mostly common pathway
Proteolytic cleavage of the hydrophobic N-terminal signal peptide sequence
Proteolytic cleavage at a site defined by pairs of basic amino acid residues
Proteolytic cleavage at sites designated by single Arg residues
Post-translational modification: C-terminal amidation, N-terminal acetylation, phosphorylation, glycosylation

54. Heptadecapeptide secreted by the antral mucosa of stomach
Gastrin as an Example
Heptadecapeptide secreted by the antral mucosa of stomach
Gastrin stimulates acid secretion in stomach
Product of preprogastrin residues
Signal peptide cleavage leaves progastrin residues
Cleavage at Lys and Arg (basic) residues and C-terminal amidation leaves gastrin
N-terminal residue of gastrin is pyroglutamate
C-terminal amidation involves destruction of Gly

58. Protein-Tyrosine Phosphatases
The enzymes that dephosphorylate Tyr
Some PTPases are integral membrane proteins
But there are also lots of soluble PTPases
Cytoplasmic PTPases have N-term. catalytic domains and C-terminal regulatory domains
Membrane PTPases all have cytoplasmic catalytic domain, single transmembrane segment and an extracellular recognition site

60. Soluble or Membrane-Bound
Guanylyl Cyclases
Soluble or Membrane-Bound
Membrane-bound GCs are the other group of single-transmembrane-segment receptors (besides RTKs)
Peptide hormones activate the membrane-forms
Note speract and resact, from mammalian ova
Activation may involve oligomerization of receptors, as for RTKs

63. Soluble Guanylyl Cyclases
Receptors for Nitric Oxide
NO is a reactive, free-radical that acts either as a neurotransmitter or as a second messenger
NO relaxes vascular smooth muscle (and is thus involved in stimulation of penile erection)
NO also stimulates macrophages to kill tumor cells and bacteria
NO binds to heme of GC, stimulating GC activity 50-fold
Read about NO synthesis and also see box on Alfred Nobel

67. Protein Modules in Signal Transduction
Signal transduction in cell occurs via protein-protein and protein-lipid interactions based on protein modules
Most signaling proteins consist of two or more modules
This permits assembly of functional signaling complexes

69. Localization of Signaling Proteins
Adaptor proteins provide docking sites for signaling modules at the membrane
Typical case: IRS-1 (Insulin Receptor Substrate-1)
N-terminal PH domain
PTB domain
18 potential tyrosine phosphorylation sites
PH and PTB direct IRS-1 to receptor tyrosine kinase - signaling events follow!

70. Signaling Pathways from Membrane to the Nucleus
The complete path from membrane to nucleus is understood for a few cases
See Figure 34.38
Signaling pathways are redundant
Signaling pathways converge and diverge
This is possible with several signaling modules on a signaling protein

73. Module Interactions Rule!
The interplay of multiple modules on many signaling proteins permits a dazzling array of signaling interactions
See Figure 34.40
We can barely conceive of the probable extent of this complexity
For example, it is estimated that there are more than 1000 protein kinases in the typical animal cell - all signals!

75. Glucocorticoids, mineralocorticoids, vitamin D and the sex hormones
Steroid Hormones
Glucocorticoids, mineralocorticoids, vitamin D and the sex hormones
May either act at nucleus or at plasma membrane
Steroids are hydrophobic and cannot diffuse freely to nucleus
Receptor proteins carry steroids to the nucleus
Steroid receptor proteins are all apparently members of a gene superfamily and have evolved from a common ancestral precursor

77. Steroid Receptor Proteins
Hydrophobic domain near C-terminus that interacts with steroid itself
Central, hydrophilic domain that binds to DNA
Central DNA-binding domains are homologous to one another, with 9 conserved Cys residues
Three pairs of Cys residues are in Cys-X-X-Cys sequences - as in Zinc-finger domains
Steroid-receptor complex may bind to DNA or to transcription factors
Thyroid hormone receptor proteins are similar

82. Extracellular Effects
of steroid hormones
Two lines of evidence: action of steroids on calcium channels and other membrane proteins and the speed of certain steroid hormone effects
Example: testosterone rapidly stimulates transport of glucose, calcium and amino acids into rat kidney cells
Several demonstrations now of tight binding of steroid probes to GABA receptor and other proteins

84. Cells of Nervous Systems
Neurons and Neuroglia (Glial Cells)
Neurons contain processes, including an axon and dendrites
Axon is covered with myelin sheath and cellular sheath, except at nodes of Ranvier
The axon ends in synaptic termini, aka synaptic knobs or synaptic bulbs
Three kinds of neurons: sensory neurons, motor neurons and interneurons

86. The source of electrical potentials in neurons
Ion Gradients
The source of electrical potentials in neurons
Nerve impulses consist of electrical signals that are transient changes in the electrical potential differences (voltages) across neuron membrane
Know resting concentrations
Learn to use the equation for actual potential difference in the box on page S-43
Difference between Nernst potential and actual potential represents a thermodynamic push

88. The Action Potential
A somewhat misleading name - it refers to the series of changes in potential that constitute a nerve impulse
Small depolarization (from -60 to -40 mV) opens voltage-gated ion channels - Na flows in
Potential rises to +30 mV, Na channels close, K channels open. K streams out, lowering potential
Action potentials flow along the axon to the synapse
Number and frequency important, not intensity

91. Voltage-Gated Na, K Channels
Clustered in Nodes of Ranvier
See Figure for Na, K channel effectors
Arrangement of Na channel in membrane is like DHP receptor in muscle
See Figure for diagram of how the channel is formed in membrane
These channels are voltage-sensitive - voltage changes cause conformational changes and gating

98. Communication at the Synapse
A crucial feature of neurotransmission
Ratio of synapses to neurons in human forebrain is 40,000 to 1!
Chemical synapses are different from electrical
Neurotransmitters facilitate cell-cell communication at the synapse
Note families of neurotransmitters in Table 34.6

99. The Cholinergic Synapse
A model for many others
Synaptic vesicles in synaptic knobs contain acetylcholine (10,000 molecules per vesicle)
Arriving action potential depolarizes membrane, opening Ca channels and causing vesicles to fuse with plasma membrane
Acetylcholine spills into cleft, migrates to adjacent cells and binds to receptors
Toxin effects: botulism toxin inhibits Ac-choline release, black widow's latrotoxin protein overstimulates

101. Two Classes of Ac-Ch Receptor
Nicotinic and muscarinic
As always, toxic agents have helped to identify and purify hard-to-find biomolecules
Nicotinic Ac-Ch receptors are voltage-gated ion channels
Muscarinic Ac-Ch receptors are transmembrane proteins that interact with G proteins
Acetylcholinesterase degrades Ac-Ch in cleft
Transport proteins and V-type H+-ATPases return Ac-Ch to vesicles - called reuptake

111. Other Neurotransmitters
Excitatory and inhibitory
Glutamate is good example: nerve impulse triggers Ca-dependent exocytosis of glutamate
Glutamate is either returned to neuron, or carried into glial cells, converted to Gln and taken back to the neuron from which it was released
See 4 types of glutamate receptors in Fig
NMDA receptor is best understood for now
Note phencyclidine (angel dust) story

112. Inhibitory Neurotransmitters
GABA and Glycine
Inhibitory Neurotransmitters
Inhibitory neurotransmitters diminish the actions of activating neurotransmitters
See Figure for glutamate degradation
Excitatory glutamate is broken down to inhibitory GABA, which is broken down to non-signals
GABA & glycine receptors are chloride channels
Glycine receptor is site of action of strychnine

118. Catecholamine Neurotransmitters
Epinephrine, norepinephrine, dopamine and L-dopa are all neurotransmitters
Synthesized from tyrosine - see Fig
Excessive production of dopamine (DA) or hypersensitivity of DA receptors produces psychotic symptoms and schizophrenia
Lowered production and/or loss of DA neurons are factors in Parkinsonism

120. Neurological Disorders
Depression, Parkinsonism, etc.
Defects in catecholamine processing are responsible for many neurological disorders
Norepinephrine and dopamine systems are keys
Breakdown of NE and DA by catechol-O-methyl transferase and monoamine oxidase
Reuptake by specific transport proteins
MAO inhibitors are antidepressants
Tricyclics - antidepressants that block reuptake
Cocaine blocks reuptake, prolongs effects of DA

121. Peptide Neurotransmitters
Lots more to learn here!
Likely to be many peptide NTs
Concentrations are low; purification is hard
Roles are complex
Endorphins and enkephalins are natural opioids
Endothelins affect smooth muscle contraction, vasoconstriction, mitogenesis, tissue changes
Vasoactive intestinal peptide stimulates AC (to make cAMP) via G proteins, and its effects are synergistic with those of other neurotransmitters

122. Sensory Transduction Similarities between sight, taste, smell
Specialized sensory cells translate stimulus into electrical signals in adjacent neurons
Vision is the paradigm system
Absorption of light quanta by rhodopsin isomerizes retinal (11-cis to all-trans)
Light is absorbed by rhodopsin in the outer segments of rod and cone cells