Chapter 34 Slides

Biochemistry 2/e - Garrett & Grisham

Chapter 34

The Reception and Transmission of Extracellular Information to accompany

Biochemistry, 2/e by

Reginald Garrett and Charles Grisham

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Copyright © 1999 by Harcourt Brace & Company


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



Outline

• 34.6 Specific Phospholipases

• 34.7 Calcium as a Second Messenger

• 34.8 Protein Kinase C

• 34.9 The Single TMS-receptor

• 34.10 Protein Modules

• 34.11 Steroid Hormones

• SPECIAL FOCUS: Neurotransmission and Sensory Systems



Classes of Hormones

(There may be others, but we doubt it...)

• Steroid Hormones - derived from cholesterolregulate 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



Signal-Transducing Receptors

Transmit Hormone Message

• Non-steroid hormones bind to plasma membrane and activate a signaltransduction pathway inside the cell

• Steroid hormones may either

– bind to the plasma membrane

– or

– enter the cell and travel to the nucleus





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

– extracellular site for hormone (ligand)

– intracellular catalytic domain - either a tyrosine kinase or guanylyl cyclase

• Oligomeric ion channels



Second Messengers

Many and there may be more!

• The hormone is the "first messenger"

• The second messenger - Ca 2+ , 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



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







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





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



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

 migrates to effector sites, activating or and inhibiting

• But it is now clear that G

 signalling device also functions as a





Signalling Roles for G(



)

A partial list

• Potassium channel proteins

• Phospholipase A

2

• Yeast mating protein kinase Ste20

• Adenylyl cyclase

• Phospholipase C

• Calcium channels

• Receptor kinases



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 G s protein is known as G i and the inhibitory G

• G i may act either by the G i

 subunit binding to

AC or by the G i

 complex complexing all the G i

 and preventing it from binding to AC

• Read about the actions of cholera toxin and pertussis toxin







The ras Gene and p21

ras

An oncogene and its product

• a gene first found in ra t s arcoma 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







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





Phospholipases Release

Second Messengers

• Inositol phospholipids yield IP

3 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



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 C

20 s, DAG and/or PA

• Sphingomyelin and glycosphingolipids also produce signals

• Ceramide (from SM) is a trigger of apoptosis - programmed cell death













Ca

2+

as a Second Messenger

Several sources of Ca 2+ in cells!

• [Ca 2+ ] 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!

• IP

3

(made by action of phospholipase C) is the trigger

• See Figures 34.17-34.19









Calcium Oscillations!

M. Berridge's model of Ca 2+ signals

• Ca 2+ was once thought to merely rise in cells to signal and drop when the signal was over

• Berridge's work demonstrates that Ca 2+ levels oscillate in cells!

• The purpose may be to protect cell components that are sensitive to high calcium, or perhaps to create waves of

Ca 2+ in the cell





Ca

2+

-Binding Proteins

Mediators of Ca 2+ effects in cells

• Many cellular proteins modulate Ca 2+ effects

• 3 main types: protein kinase Cs , Ca 2+ modulated proteins and annexins

• Kretsinger characterized the structure of parvalbumin , prototype of Ca 2+ -modulated proteins

• "EF hand" proteins bind BAA helices







Transduction of two second messenger signals

PKC is activated by DAG and Ca 2+

• Most PKC isozymes have several domains, including ATP-binding domain, substrate-binding domain, Ca-binding domain and a phorbol esterbinding domain

• Phorbol esters are apparent analogues of DAG

• Cellular phosphatases dephosphorylate target proteins

• Read about okadaic acid











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 pp60 v-src was first known

• Two posttranslational modifications







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





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



Proteolytic Processing

A mostly common pathway

• Proteolytic cleavage of the hydrophobic Nterminal 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



Gastrin as an Example

Heptadecapeptide secreted by the antral mucosa of stomach

• Gastrin stimulates acid secretion in stomach

• Product of preprogastrin - 101-104 residues

• Signal peptide cleavage leaves progastrin -

80-83 residues

• Cleavage at Lys and Arg (basic) residues and Cterminal amidation leaves gastrin

• N-terminal residue of gastrin is pyroglutamate

• C-terminal amidation involves destruction of Gly









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





Guanylyl Cyclases

Soluble or Membrane-Bound

• Membrane-bound GCs are the other group of single-transmembrane-segment receptors (besides RTKs)

• Peptide hormones activate the membraneforms

• Note speract and resact, from mammalian ova

• Activation may involve oligomerization of receptors , as for RTKs







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









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





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!



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







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!





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





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











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





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 i nterneurons





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





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







Voltage-Gated Na, K

Channels

Clustered in Nodes of Ranvier

• See Figure 34.52 for Na, K channel effectors

• Arrangement of Na channel in membrane is like

DHP receptor in muscle

• See Figure 34.55 for diagram of how the channel is formed in membrane

• These channels are voltage-sensitive - voltage changes cause conformational changes and gating















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



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





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





















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. 34.68

• NMDA receptor is best understood for now

• Note phencyclidine (angel dust) story



GABA and Glycine

Inhibitory Neurotransmitters

• Inhibitory neurotransmitters diminish the actions of activating neurotransmitters

• See Figure 34.70 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













Catecholamine

Neurotransmitters

• Epinephrine, norepinephrine, dopamine and

L-dopa are all neurotransmitters

• Synthesized from tyrosine - see Fig. 34.72

• 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





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



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



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


Chapter 34 Slides

Chapter 34

Outline

Outline

Classes of Hormones

Signal-Transducing Receptors

Transmit Hormone Message

Types of Receptors

Second Messengers

cAMP and Glycogen

Phosphorylase

How are the hormone receptor and AC coupled?

G Proteins

Heterotrimeric G Proteins

Signalling Roles for G(

)

Stimulatory and Inhibitory G

The ras Gene and p21

7-TMS Receptors

Phospholipases Release

Second Messengers

Other Lipids as Messengers

Ca

as a Second Messenger

Calcium Oscillations!

Ca

-Binding Proteins

Transduction of two second messenger signals

Single TMS Receptors

Receptor Tyrosine Kinases

The Polypeptide Hormones

Proteolytic Processing

Gastrin as an Example

Protein-Tyrosine Phosphatases

Guanylyl Cyclases

Soluble Guanylyl Cyclases

Protein Modules in Signal

Transduction

Localization of Signaling

Proteins

Signaling Pathways from

Membrane to the Nucleus

Module Interactions Rule!

Steroid Hormones

Steroid Receptor Proteins

Extracellular Effects

Cells of Nervous Systems

Ion Gradients

The Action Potential

Voltage-Gated Na, K

Channels

Communication at the

Synapse

The Cholinergic Synapse

Two Classes of Ac-Ch Receptor

Other Neurotransmitters

GABA and Glycine

Catecholamine

Neurotransmitters

Neurological Disorders

Peptide Neurotransmitters

Sensory Transduction

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