Lecture 3

Question 1

signal transduction controls what?

Answer 1

cell function

Question 2

Endocrine (aka hemocrine)

Answer 2

Ligand is generated by a cell, released into circulation (enters blood via capillary fenestrations) where it affects a distant target tissue

ex: endocrine hormones (insulin, ACTH, etc)

Question 3

Paracrine

Answer 3

-involves the extrusion of biomolecules into the surrounding interstitial spaces and contacts nearby tissue or “neighbor” (coordinates activity of a tissue)
-Delivers high concentration of biomolecule to target site.

ex: Prostaglandins

Question 4

Intercrine

Answer 4

direct paracrine transfer of cytoplasmic messenger molecule into adjacent cells via specialized gap junctions

Question 5

Neurocrine

Answer 5

-release of peptide hormones, neurotransmitters or neuromodulators by neurons
-Subcategories include: 1) synaptic or 2) non-synaptic (aka neurosecretion)
-messenger is transported to its local or distal site of action by ECF or blood.

Question 6

Solinocrine

Answer 6

secretion of messenger occurs into a “hollow tube”

ex: ductal structures, including GI lumen, bronchi, urogenital tract

Question 7

Juxtacrine

Answer 7

-Cell-cell contact
– Messenger molecule does not traverse a fluid phase to reach another cell but remains associated with the plasma membrane of the signaling cell while acting on an immediately adjacent cell
-Long-term modulator of tissue behavior.

ex: Integrins bind neutrophils and allow for transmigration across endothelial cells

Question 8

Autocrine

Answer 8

-Secreted molecule interacts with same cell that produced it, i.e. direct feedback on cell that released the signal.

ex: Interleukin-2 (IL-2) is released from activated T lymphocytes and can either increase or decrease its own release

Question 9

Most transmembrane signaling is accomplished by only a few different molecular mechanisms:

Answer 9

*receptors on the cell surface
*intracellular receptors (that are not enzymes)
*enzymes

Question 10

what are the 5 Signaling Mechanisms and Drug Action

Answer 10

1. A lipid-soluble ligand that crosses the membrane and acts on an intracellular receptor.
2. a transmembrane receptor protein whose intracellular enzymatic activity is allosterically regulated by a ligand that binds to a site on the protein’s extracellular domain.
3. a transmembrane receptor that binds and stimulates a protein tyrosine kinase
4. a ligand-gated transmembrane ion channel that can be induced to open or close by the binding of a ligand
5. a transmembrane receptor protein that stimulates a GTP-binding signal transducer protein (G protein), which in turn generates an intracellular second messenger.

Question 11

Lipid-soluble chemical crosses on what and acts as what?

Answer 11

crosses on the plasma membrane and acts on an intracellular receptor

*which may be an enzyme or a regulator of gene transcription

Question 12

example of intracellular receptors for lipid soluble agents

Answer 12

nitric oxide (NO) is a freely diffusible gas which acts by crossing the membrane and stimulating an intracellular enzyme Guanylyl Cyclase

Question 13

examples of lipid-soluble ligands

Answer 13

Corticosteroids, mineralocorticoids, sex steroids,
Vitamin D and thyroid hormone

anything that has an “oids” at the end

Question 14

Intracellular receptors for these agents bind their ligands and undergo a conformational change that allows what?

Answer 14

allows them to enter the nucleus and help regulate gene expression

Question 15

Cell nucleus does what?

Answer 15

Nuclear signaling;
Transcription of genes

Question 16

Binding of glucocorticoid hormone to its normal receptor relieves what?

Answer 16

an inhibitory constraint, HSP90– usually keeps the receptor in the cytoplasm.

Question 17

Binding of hormone to the ligand-binding domain triggers what?

Answer 17

release of hsp90

*This allows the folding of the DNA-binding domains and transcription-activating domains of the receptor to fold into their proper conformations so that the activated receptor can initiate transcription of target genes in the nucleus.

Question 18

The Mechanism Used by Hormones that act by regulating gene expression has two therapeutically important consequences

Answer 18

1. Hormones produce their effects after a characteristic lag period of 30 minutes to several hours the time required for the synthesis of new proteins
2. Effects of these agents can persist for hours or days after the agonist has been removed.

Question 19

Ligand-Regulated Transmembrane Enzymes (Including Receptor Tyrosine Kinases)

Answer 19

know this slide

Question 20

For Ligand-Regulated Transmembrane Enzymes, this class of receptor molecules mediates the first steps in signaling by

Answer 20

insulin

epidermal growth factor (EGF)

platelet-derived growth factor

atrial natriuretic factor (ANF)

transforming growth factor-beta

Question 21

HORMONEs will bind to the extracellular hormone binding domains resulting in a what change?

Answer 21

conformational change in the receptors which will cause them to associate in pairs (dimerize)

Question 22

what is AUTOphosphorylation?

Answer 22

TYROSINE residues in both cytoplasmic domains become phosphorylated. Each domain is believed to be phosphorylated by the other in a cross-phosphorylation event

Question 23

After autophosphorylation, receptors catalyze phosphorylation of tyrosine residues on different

Answer 23

downstream signaling proteins

Question 24

know this slide

Answer 24

Question 25

what is one example of complexity?

Answer 25

Each of the growth factors initiates in its specific target cells a complex program of cellular events ranging from altered membrane transport of protons, other ions, and metabolites to characteristic changes in the expression of many genes.

Question 26

Insulin uses a single class of receptors that:

Answer 26

*to trigger increased uptake of glucose and amino acids *and to regulate the metabolism of glycogen and triglycerides in the cell.

Question 27

The tyrosine kinase receptors provide attractive targets for what?

Answer 27

Drug Development

Question 28

it is easy to envision therapeutic uses for

Answer 28

specific inhibitors of growth factor receptors, especially in neoplastic disorders -No dimerization -No receptor phosphorylation -No phosphorylation of Downstream substrates

Question 29

The intensity and duration of action of EGF, PDGF and other agents that act via this class of receptors are

Answer 29

limited by receptor down-regulation

Question 30

Ligand binding induces accelerated ________ of receptors from the cell surface

Answer 30

endocytosis

Question 31

The TOTAL number of cell-surface receptors is reduced (down regulated) and the cell’s responsiveness to ligand is

Answer 31

diminished

Question 32

Receptor Binding & Activation

Answer 32

Ligand binds to its receptor

Question 33

Coated Pit Formation

Answer 33

Clathrin forms cage around forming endosome

Question 34

Clathrin Vesicle Formation

Answer 34

Vesiculation occurs

Question 35

Vesicle Uncoating

Answer 35

Clathrin coat is removed

Question 36

CURL Endosome Forms

Answer 36

Compartment of unbinding of receptor and ligand

Question 37

T/F: Receptor recycles to the surface or is sent to a digestive vacuole

Answer 37

dis bish true

Question 38

What Happens to Receptors After They Are Internalized

Answer 38

Can lead to receptor downregulation

Question 39

Ligand-Regulated Transmembrane Enzymes (Including Receptor Serine/Threonine Kinases)

Answer 39

Certain regulators of growth and differentiation act through transmembrane receptors that have cytoplasmic domains with serine/thr kinase activity (eg. transforming growth factor-beta) TGF-beta

Question 40

Signaling Pathway of TGF-b

Answer 40

TGF-b regulates growth and proliferation of cells, blocking the growth of many different cell types. The TGF-b receptor includes Type I and Type II subunits that are serine-threonine kinases that signal through the Smad family of proteins. Binding of transforming growth factor b (TGF-b) to its cell surface receptor Type II leads to the phosphorylation of the Type I receptor by Type II. The Type I receptor is then able to phosphorylate and activate the Smad2 protein. Smad2, in combination with Smad4, is translocated to the nucleus where the activated Smad complex recruits other transcription factors (TF) that activate the expression of target genes

Question 41

Ligand-Regulated Transmembrane Enzymes (Guanylyl cyclases )

Answer 41

Atrial natriuretic factor (ANF), an important regulator of blood volume and vascular tone, acts on a transmembrane receptor whose intracellular domain has a guanylyl cyclase activity (GC). Like receptor tyrosine kinases, and receptor serine kinases, ANF receptors are active in their dimeric forms.

Question 42

Cytokines include:

Answer 42

A vast array of relatively low molecular weight, pharmacologically active proteins that are secreted by the cell to alter either its functions (autocrine effect) or those of adjacent cells (paracrine effect).

Question 43

Cytokine receptors respond to

Answer 43

a heterogeneous group of peptide ligands that includes: growth hormone erythropoietin interferons other regulators of growth and regulation

Question 44

Cytokine Receptors are activated by ligand to form

Answer 44

a dimer

Question 45

Janus Kinases (JAK) bind

Answer 45

noncovalenty to the cytoplasmic side of the cytokine receptor

Question 46

The JAK kinases now become active and phosphorylate

Answer 46

tyrosine residues on the receptor. This is followed by the recruitment of another type of protein called STATs (signal transducers and activators of transcription)

Question 47

Recruitment of the STATs occurs at the

Answer 47

phosphorylated tyrosines.

Question 48

JAKs then perform a second phosphorylation on tyrosines, this time on the STATs

Answer 48

The phosphorylated STATs dissociate from the cytoplasmic side of the receptor and form a dimer with each other.

Question 49

The phosphorylated STATs dissociate from the cytoplasmic side of the receptor and

Answer 49

form a dimer with each other

Question 50

The STAT dimer migrates into the nucleus and

Answer 50

modulates gene transcription

Question 51

The multistep signaling process provides several attractive targets for potential drugs:

Answer 51

*cytokine binding site *1st phosphorylation *2nd phosphorylation *Nuclear translocation

Question 52

Many drugs in clinical medicine act by

Answer 52

mimicking or blocking the actions of endogenous ligands that regulate the flow of ions across the plasma membrane

Question 53

The natural ligands of these receptors include

Answer 53

acetylcholine gamma-aminobutyric acid excitatory amino acids (glycine, aspartate, glutamate) Each of these receptors transmits its signal across the plasma membrane by increasing transmembrane conductance of the relevant ion thereby altering the electrical potential across the membrane

Question 54

TOP VIEW of the Nicotinic Acetylcholine Receptor

Answer 54

*a look from the extracellular space. The channel opens into the cytoplasm In the absence of ligand, the pentameric nicotinic acetylcholine receptor is closed and does not allow the charged sodium ion passage into the cytoplasm

Question 55

Acetylcholine binding (represented by the white spheres on the alpha subunits), induces a conformational change in the receptor

Answer 55

A channel opens allowing passage of the ion. This particular receptor regulates passage of sodium ions. Na+ can now flow into the cell.

Question 56

G protein receptors are called what?

Answer 56

Serpentine’ receptors because the receptor polypeptide chain resembles a snake. G-protein coupled receptors are structurally-related. Often called 7 transmembrane spanning receptors.

Question 57

G-protein coupled receptors include receptors for

Answer 57

adrenergic amines serotonin, histamine acetylcholine (muscarinic) many peptide hormones odorants photons (rods and cones)

Question 58

T/F: Similarities between amino acid sequences of serpentine receptors suggest that all of these receptors share a common evolutionary precursor

Answer 58

True

Question 59

Many extracellular ligands act by increasing the intracellular concentrations of 2nd messengers such as

Answer 59

*adenosine-3’,5’-monophosphate (cAMP) *calcium ions *phosphoinositides

Question 60

In most cases, these ligands use a transmembrane signaling system with three separate components

Answer 60

1. extracellular ligand is specifically detected by a cell surface receptor 2. the receptor activates a G protein (cytoplasmic face) 3. activated G protein changes the activity of an effector element (eg. enzyme like adenylyl cyclase)

Question 61

Increased Effector Activity

Answer 61

this is an example: increased adenylyl cyclase activity… increased cAMP in cell

Question 62

The G protein that activates adenylyl cyclase is

Answer 62

Gs

Question 63

Gs is activated by what?

Answer 63

a host of hormones and neurotransmitters, each of which acts via a specific receptor

Question 64

This is a partial list of endogenous ligands that increase intracellular cAMP levels via Gs:

Answer 64

Adrenocorticotropic hormone Catecholamines (Beta adrenoceptors) Follicle Stimulating Hormone Glucagon Histamine (H2 receptors)

Question 65

Gs and other G proteins use a molecular mechanism that involves what?

Answer 65

binding and hydrolysis of GTP

Question 66

When the G-protein coupled receptor is not activated by ligand

Answer 66

the G-protein is associated with GDP

Question 67

GDP= guanosine diphosphate

Answer 67

inactive

Question 68

GTP= guanosine triphosphate

Answer 68

active

Question 69

what happens when the G-protein coupled receptor is activated by ligand

Answer 69

GDP is released and GTP enters into the nucleotide binding site of the G protein

Question 70

Activated G-protein (bound to GTP) migrates from the receptor and regulates the activity of an

Answer 70

effector enzyme or ion channel (E)

Question 71

T/F: Hydrolysis of GTP terminates the G protein signal

Answer 71

true *The signal is terminated by the hydrolysis of GTP

Question 72

when G protein is bound again to GDP, what happens

Answer 72

couples with the receptor and awaits activation

Question 73

know this

Answer 73

Question 74

A neurotransmitter like norepinephrine (NE) may encounter its membrane receptor for a short time

Answer 74

a few milliseconds

Question 75

When the Gs protein coupled to the NE receptor binds GTP and becomes activated, it may remain active for _____________ enormously amplifying the original signal.

Answer 75

tens of seconds

Question 76

Gs receptors

Answer 76

Receptors for : Beta-adrenergic amines glucagon histamine serotonin other hormones *INCREASES ADENYL CYCLASE (activity) and increased cAMP

Question 77

Gi (1-3)

Answer 77

Receptors for: alpha2-adrenergic amines acetylcholine (muscarinic) opioids serotonin others *Decreases ADENYL CYCLASE (activity) and decrease cAMP

Question 78

Gq

Answer 78

Receptors for: acetylcholine (muscarinic) serotonin (5-HT1C) many others *Increases phospholipase C (activity) and increases IP3, DAG, cytoplasmic Ca++

Question 79

what happens after ligand binding in G receptors

Answer 79

conformational change in the receptor is sensed by the cytoplasmic loops and G-protein is activated (GDP exchanged for GTP).

Question 80

what happens after After hydrolysis of GTP?

Answer 80

G protein/GDP is inactive and couples again with the receptor

Question 81

Cytoplasmic tail of the G-protein coupled receptor contains what?

Answer 81

numerous serine and threonine residues These residues can be phosphorylated

Question 82

Phosphorylation may be associated with

Answer 82

diminished receptor/G-protein interaction

Question 83

Receptor-mediated responses to drugs and hormonal agonists often

Answer 83

desensitize with time (signal is there but no response)

Question 84

After reaching an initially high level, the response (eg. intracellular cAMP accumulation, Na+ influx, contractility, etc..)

Answer 84

gradually diminishes over seconds or minutes, even in the continued presence of the agonist

Question 85

T/F: This ‘desensitization’ is usually not reversible

Answer 85

False it is reversable 15 min after removal of the agonist, a second exposure to agonist results in a response similar to the initial response

Question 86

what is desensitization?

Answer 86

even though the receptors are saturated with agonist, the response gradually decreases.

Question 87

Although many kinds of receptors undergo desensitization, the mechanism is

Answer 87

obscure in most cases (eg. the nicotinic acetylcholine receptor)

Question 88

what is the exception of receptor regulator mechanisms?

Answer 88

in the case of the beta-adrenoceptor, the mechanism has been worked out in some detail

Question 89

Ligand binding activates Gs-GTP as well as a kinase called

Answer 89

BARK (beta-adrenergic receptor kinase) BARK phosphorylates the cytoplasmic tail of the receptor on serine and threonine residues

Question 90

beta-arrestin does what

Answer 90

binds to the cytoplasmic tail, which results in a diminished ability of the receptor to interact with the G protein; reducing the response to agonist.

Question 91

Upon removal of agonist

Answer 91

*cellular phosphatases cleave the phosphates from the tail *BARK activity is decreased (stops phosphorylating the cytoplasmic tail of the receptor) The receptor is then ready to fully respond to agonist; it has ‘re-set itself..’

Question 92

GPCR is activated by

Answer 92

ligand

Question 93

The G protein has become activated by

Answer 93

GTP binding

Question 94

In addition to G-protein activation, receptor activation also stimulates

Answer 94

the BARK kinase to bind to the cytoplasmic tail of the receptor

Question 95

Activated BARK kinase phosphorylates

Answer 95

serine and threonine residues in the tail

Question 96

Phosphorylation of the tail increases the affinity for

Answer 96

beta-arrestin, which then binds to the tail

Question 97

Hydrolysis of the G protein GTP inhibits activation of effector (E) BUT..

Answer 97

The G protein/GDP cannot couple with the third intracellular loop because the site is hindered by beta-arrestin. Even though the ligand is bound, the response will decrease because the Gprotein-GDP cannot be activated

Question 98

When the agonist dissociates from receptor

Answer 98

*BARK is inactivated *the phosphates on the cytoplasmic tail are cleaved by cellular phosphatases

Question 99

what are phosphates?

Answer 99

enzymes which cleave phosphate groups off of proteins

Question 100

agonist not bound to receptor

Answer 100

*Inorganic phosphates are released (Pi) *Beta-arrestin dissociates from the cytoplasmic tail *G-protein/GDP can once again couple with the receptor– the receptor has ‘re-set’ itself!

Question 101

receptor desensitization differs from the slower onset and more prolonged effect of receptor downregulation

Answer 101

An agonist- induced decrease in receptor number through internalization and degradation