1. CELL SIGNALING AND MOTILITY (BIOL 3373)
Lecture 3

2. WHAT IS The CELL SIGNALING?
How cells receive and respond to signals from their surroundings
On one hand, cell signaling regulates gene expression and controls the cell fate (proliferation, motility, differentiation and programmed cell death, or apoptosis).
On the other hand, cell signaling allows for the organization of cells into tissues, which, in turn, generate organs. In addition, cell signaling is essential for the maintenance of cells, tissues and organs.

3. CELL SIGNALING
Communication among cells is referred as intercellular signaling.
Cells communicate with each other through signaling molecules.
Signaling molecules could be:
proteins, small peptides, amino acids,
nucleotides,
steroids, retinoids, fatty acid derivatives,
nitric oxide, carbon monoxide

4. Forms of signaling molecules : Steroid hormones
Steroid hormones are a group of hormones that belong to the class of chemical compounds known as steroids. All steroid hormones are derived from cholesterol. They are transported through the bloodstream to the cells of various target organs where they carry out the regulation of a wide range of physiological functions. These hormones often are classified according to the organs that synthesize them.
The adrenal cortex produces the adrenocortical hormones, which consist of the glucocorticoids and the mineralocorticoids. Glucocorticoids such as cortisol control many metabolic processes, including the formation of glucose and the deposition of glycogen in the liver.
Mineralocorticoids such as aldosterone help maintain the balance between water and salts in the body, predominantly exerting their effects within the kidney.
The androgens are the male sex hormones, responsible for the development and maintenance of reproductive function in the male. The principal androgen, testosterone, is produced by the testes. Estrogens are female sex hormones. They are secreted mainly by the ovaries.
Estradiol is the most potent of the estrogens. the estrogens promote the development of the primary and secondary female sex characteristics.
Progestins, the most important of which is progesterone, are the other type of female sex hormone and are named for their role in maintaining pregnancy (pro-gestation).

5. Forms of signaling molecules- Gas - NO
Nitric oxide (NO) is a gas molecule produced and released by endothelial cells (signaling cells) and rapidly diffuses across the membranes.
NO binds to an enzyme inside the smooth muscle cells (target cell), inducing cell relaxation

6. Forms of signaling molecules
Neurotransmitters
Neurotransmitters are chemicals produced and released by neurons.
Neurotransmitters travel across the synapse and allow communication between neurons.

7. Forms of signaling molecules
Neurotransmitters

8. Forms of signaling molecules
Peptide Hormones and Growth Factors

9. CELL SIGNALING
Cells that produce and release the signaling molecules are signaling cells.
Cells that receive the signal are target cells
Targets cells posses specific receptors that recognize signaling molecules.
signaling cell
target cell
signaling molecules
receptor

10. CELL SIGNALING
Depending on the distance that the signaling molecule has to travel, we can talk about four types of signaling:
Contact- dependent signaling requires cells to be in direct membrane-membrane contact.
The signaling molecule remains bound to the surface of the signaling cells. It influences only cells (target cells) contact it via a specific protein receptor.
Contact-dependent signaling is especially important during development and immune response
from Alberts et al., Molecular Biology of the Cell. 6th edition.

11. In paracrine signaling
CELL SIGNALING
In paracrine signaling
the signaling molecules are local mediators and affects only target cells in the proximity of the signaling cell. An example is the conduction of an
electric signal from one nerve cell to another or to a muscle cell. In this case the signaling molecule is a neurotransmitter.
from Alberts et al., Molecular Biology of the Cell. 6th edition.

12. CELL SIGNALING
In autocrine signaling cells respond to molecules they produce themselves. So signaling cells are also target cells.
Examples include cancer cells as they can produce signals that stimulate their own survival and proliferation.
autocrine signaling
signaling cell = target cell
from Alberts et al., Molecular Biology of the Cell. 6th edition.

13. CELL SIGNALING
Large multicellular organisms use long- range signaling that coordinate the behavior of the cells in remote part of the body.
Synaptic signaling is performed by neurons that transmit signals electrically along their axons and release neurotransmitters at synapses, which can be located far away from the neuron cell body.
from Alberts et al., Molecular Biology of the Cell. 6th edition.

14. CELL SIGNALING
In endocrine signaling signaling molecules (hormones) are produce by endocrine cells and sent through the blood stream to distant cells

15. The Three Stages of Cell Signaling
Earl W. Sutherland discovered how the hormone epinephrine acts on cells
Sutherland suggested that cells receiving signals went through three processes:
Reception
Transduction
Response

16. Relay molecules in a signal transduction pathway
Overview of cell signaling
Cell signaling consists of 3 stages
EXTRACELLULAR
FLUID
Receptor
Signal
molecule
Relay molecules in a signal transduction pathway
Plasma membrane
CYTOPLASM
Activation
of cellular
response
Reception 1
Transduction 2
Response 3

17. Three Stages of Cell Signaling: 1 Reception
EXTRACELLULAR
FLUID
CYTOPLASM
Plasma membrane 1 1
Reception
The receptor and signaling molecules fit together (lock and key model)
Receptor
Signaling
molecule
Signaling molecule binds to the receptor protein at the cell surface 17

18. 1- Reception: A signal molecule binds to a receptor protein, causing it to change shape
The binding between a signal molecule (ligand) and receptor is highly specific
A conformational change in a receptor is often the initial transduction of the signal
Most signal receptors are plasma membrane proteins 18

19. Receptors in the Plasma Membrane

20. Intracellular Receptors
Some receptor proteins are intracellular, found in the cytosol or nucleus of target cells
Small or hydrophobic chemical messengers can readily cross the membrane and activate receptors
Examples of hydrophobic messengers are the steroid and thyroid hormones of animals
An activated hormone-receptor complex can act as a transcription factor, turning on specific genes

21. Hydrophobic signaling molecules can cross the plasma membrane and bind to nuclear receptors

22. Hormone
(testosterone)
EXTRACELLULAR
FLUID
The steroid
hormone testosterone
passes through the
plasma membrane.
Plasma
membrane
Testosterone binds
to a receptor protein
in the cytoplasm,
activating it.
Receptor
protein
Hormone-
receptor
complex
The hormone-
receptor complex
enters the nucleus
and binds to specific
genes.
DNA
mRNA
The bound protein
stimulates the
transcription of
the gene into mRNA.
NUCLEUS
New protein
The mRNA is
translated into a
specific protein.
CYTOPLASM

23. The nuclear receptor superfamily
Nuclear receptors are inactive without signaling molecules (ligands)
When the nuclear receptor interacts with the specific ligand, it becomes activate and induces the transcription of target genes

24. Three Stages of Cell Signaling: 2 Transduction
EXTRACELLULAR
FLUID
CYTOPLASM
Plasma membrane 1 1
Reception 2
Transduction
Receptor
2nd Messenger!
Relay molecules in a signal transduction pathway
Signaling
molecule
The signal from the receptor is converted into a intracellular message that produces a cellular response. 24

25. Includes a NETWORK of molecular and cellular events
SIGNAL TRANSDUCTION: The study of the molecular circuits responsible for the generation of a cellular response after the delivery of a signal.
Includes a NETWORK of molecular and cellular events
conveying a SIGNAL from the outside to the inside of the cell.

26. SIGNAL TRANSDUCTION
Signaling molecules (ligands) bind to receptors on target cells.
After the binding with the ligand, the receptor activates one or more intracellular signaling within the target cells, that involves several proteins ( transducer Protein).
The intracellular signaling modulates the activity of target proteins (also known as effector proteins) thereby the behavior of the cells.
from Alberts et al., Molecular Biology of the Cell. 6th edition.

27. Three Stages of Cell Signaling: 3 Response
EXTRACELLULAR
FLUID
CYTOPLASM
Plasma membrane 1
Reception 2
Transduction 3
Response
Receptor
Activation
of cellular
response
Relay molecules in a signal transduction pathway
Signaling
molecule
Cellular responses can be different and complex (i.e. change in gene expression, cell motility, cell growth, cell differentiation, cell death), depending on cell types. 27

28. Cytoplasmic and Nuclear Responses
The response may occur in the cytoplasm or in the nucleus
Many signaling pathways regulate the synthesis of enzymes or other proteins, usually by turning genes on or off in the nucleus
The final activated molecule in the signaling pathway may function as a transcription factor

29. Cytoplasmic and Nuclear Responses
Many other signaling pathways regulate the synthesis of enzymes or other proteins, usually by turning genes on or off in the nucleus
The final activated molecule may function as a transcription factor

30. Fine-Tuning of the Response
There are four aspects of fine-tuning to consider
signal Amplification (and thus the response)
Specificity of the response,
Efficiency of response,
Initiation of the signal,
Termination of the signal,

31. Signal Amplification
At each step of many signal transduction pathways, the number of activated participants in the pathway increases. This is referred as signal amplification,
For example, one epinephrine-activated GPCR activates 100s of Gas-GTP complexes, which in turn activate 100s of adenylyl cyclase molecules, that each produce hundreds of cAMP molecules, and so on. The overall amplification associated with epinephrine signaling is estimated to be ~108-fold (figure 1)
figure 1

32. Specificity of the response
Different kinds of cells have different collections of proteins
These differences in proteins give each kind of cell specificity in detecting and responding to signals
The response of a cell to a signal depends on the cell’s particular collection of proteins
Pathway branching and “cross-talk” further help the cell coordinate incoming signals

33. Specificity of the response
Signal
molecule
Receptor
Relay
molecules
Response 1
Response 2
Response 3
Cell B. Pathway branches,
leading to two responses
Cell A. Pathway leads
to a single response
Cell C. Cross-talk occurs
between two pathways
Response 4
Response 5
Activation
or inhibition
Cell D. Different receptor
leads to a different response

34. The same signal can induce different responses in distinct cell types

35. Efficiency of response depends on:
The expression of specific receptors;
The bioavailability of transducer molecules:
Expression levels/half-life
Localization within the cell
Modality of activation/inactivation
The bioavailability of effector molecules:
cytoskeletal elements (morphological changes)
transcription factors (changes in gene expression)
proteolytic enzymes (cell death)

36. INITIATION of Signal: RECEPTOR ACTIVATION/ InACTIVATION
For EACH signaling pathway there is a common feature that defines a sequence of events:
The receptor is in an INACTIVE STATE in the absence if the signal
When the signal arrives, it BINDS to the receptor, which MODIFIES its conformation
This change in the receptor ACTIVATES other molecules (i.e. a downstream signaling cascade)

37. Termination of the Signal:
Inactivation mechanisms are an essential aspect of cell signaling
When signal molecules leave the receptor, the receptor reverts to its inactive state.

38. THE SIGNALING PATHWAY VIA G-PROTEIN

39. G-protein coupled receptors
G- protein coupled receptors consists of 3 components:
trans-membrane receptors (GPCRs) known also as serpentine receptors.
2. G proteins: Guanine nucleotide-binding proteins, trimeric G protein.
3. Effectors: Effectors can be different : adenylyl cyclase or Phosphodiesterase (PDE) or phospholipase C-β (PLC- β) or ion Channel.
Receptor
adenylyl cyclase
PDE
ion Channel
PLC- β
G protein
Effector

40. These effectors in turn regulate the intracellular concentrations of secondary messengers, such as cAMP, cGMP, diacylglycerol (DG), IP3, sodium (Na+), potassium (K+) or calcium cations (Ca2+), which ultimately lead to a physiological response, usually via the downstream regulation of gene transcription.
adenylyl cyclase
PDE
ion Channel
PLC- β
cGMP
cAMP
Na+,
Ca2+ K+ DG
IP3
Second messengers

41. G-protein coupled receptors
Several types of receptors are associated with G proteins, such as hormones, neurotransmitters, growth factors, glycoproteins, cytokines, odorants and photons

42. G-protein-coupled receptors
Transmembrane
helix C
-Terminal chain
G-Protein
binding region
Variable
intracellular loop
Extracellular
loops
Intracellular loops N
HO2C
NH2
VII VI V IV
III II I
Membrane
Single protein composed of 7 transmembrane domains with an extracellular amino terminus and an intracellular carboxyl terminus.
Intracellular C terminus changes conformation in response to a stimulus and this results in changes in the ability to recruit G proteins.

43. G-protein G proteins are composed by 3 subunits, α,β and γ.
The α subunit contains the GDP binding site as well as the GTP hydrolysis domain.

44. ADENYLYL CYCLASE : EFFECTOR OF GPCR
First messenger
(signal molecule
such as epinephrine)
Adenylyl
cyclase
G protein
G-protein-linked
receptor
GTP
ATP
Second
messenger
cAMP
Adenylyl cyclase is a multipass transmembrane protein that converts ATP to form cyclic AMP (cAMP).
Protein
kinase A
Cellular responses

45. Cyclic AMP (cAMP)
ATP
Cyclic AMP
AMP
Adenylyl cyclase
Pyrophosphate P i
Phosphodiesterase
H2O
The Adenylyl Cyclase catalyzes the reaction that leads to the production of cAMP.
cAMP is synthesized from ATP through a cyclization reaction that removes two phosphate group as pyrophosphate.
cAMP is an unstable molecule because it is soon hydrolyzed to AMP by specific Phosphodiesterases.

46. Activation of G-protein
The binding of the ligand to the receptor changes the receptor conformation.
The new receptor conformation allows the binding of the G protein.
When the G protein assembles to the receptor it alters its own conformation, therefore the GDP binding site within the α subunit is distorted and GDP is released.
Ligand 1 2 3 ß a g
Receptor G
Protein
Cell membrane ß a g ß
Binding site for G-protein opens g a
GDP
GTP

47. Activation of G-protein α subunit changes conformation
4. The binding of GTP to the α subunit promotes the closure of the nucleotide binding site within the α subunit.
5. Therefore the α subunit changes its conformation and
6. separates from both the β-γ dimer and the receptor.
This process is active while the ligand is bound to the receptor
One ligand can activate several G protein: Signal amplification 4 5 6 ß g ß ß g
GTP binds g
Fragmentation
and release a a a
GTP
α subunit changes conformation

48. Activation of G-protein
7. The α-GTP subunit interacts with the Adenylyl cyclase
8. Adenylyl cyclase becomes active and converts ATP in cAMP.
9. The cycle is completed when GTP is hydrolyzed to GDP within the α subunits. This causes the re-association of α subunit with β-γ dimer and the binding of G-protein to the receptor, which terminates the cycle.
a Subunit recombines with b-g dimer
to reform inactive G protein.
Active site
(closed)
Binding site
for a subunit
cyclic AMP
ATP
Binding
(open) P
GTP hydrolysed
to GDP catalysed
by a subunit
as-subunit
Adenylyl cyclase
GTP
GDP
Signal
transduction
(con) 7 8 9

49. Activation of protein Kinase A (PKA)
cyclic AMP
ATP
Adenylate
cyclase
The target proteins of cAMP vary depending on cell types, however the best characterized cAMP target is the PROTEIN KINASE A (PKA).
PKA phosphorylates serine or threonine residues on target proteins thereby regulating their activity.
Among the target proteins of PKA the phosphodiesterases are responsible to lower cAMP concentration thus keeping PKA activation short and localized.
PKA
Activation
Enzyme
(inactive) P
Enzyme
(active) P
Target
Protein

50. Target Proteins of protein Kinase A (PKA)
Examples of a signaling pathway mediated by cAMP-PKA include the activation of transcription regulator, the CRE binding protein (CREB).
PKA phosphorylates CREB on a single serine.
Active phosphorylated CREB (p-CREB) recruits the coactivator CREB-binding protein (CBP) (not known) to the regulatory sequence (CRE) present in many genes (i.e. somatostatin gene) that are activated by cAMP.
CREB signaling plays an important role in the learning and memory process in the brain.

51. Activation of protein Kinase A (PKA)
PKA is a complex protein consisting of 2 regulatory subunits and two catalytic domains.
The regulatory domains bind to the cytoskeleton or membrane organelle, thereby tethering PKA in specific subcellular compartment.
Four cAMP molecules bind to the PKA regulatory domains, causing their dissociation from the protein complex and the consequent activation of the catalytic subunits.

52. Double Click on box below for video

53. Olfactory receptor neurons
Smell depends on GPCRs that regulate cyclic-nucleotide-gated ion channels
Olfactory receptor neurons line on the nose.
These cells use GPCRs called olfactory receptors to recognize odors; the receptors are displayed on the modified cilia that extend from each cell.
Olfactory receptor neurons

54. Sensory transduction in olfaction
Odorant molecules bind to olfactory receptors activating a G protein, which in turn activates adenylyl cyclase . Second-messenger systems are activated, Na+ (sodium) and Ca2+ (calcium) or Ca2+ channels are opened, and the cilia are depolarized. This depolarization iniziates a nerve implulse thart travels alomg its axon to the brain

55. Vision depends on GPCRs that regulate cyclic-nucleotide-gated ion channels

56. Photo- transduction in photoreceptor cells
GMP
Light
Na+
cGMP gated ion channel
Rod cells
Photo- transduction in photoreceptor cells
In the retina the rod cells contains a pigment protein, rhodopsin, which is a GPCRs associated with the G protein, Transducin.
Signal = light
G-protein coupled receptor= Rhodopsin (Rods)
G protein = transducin
enzymatic activity = phosphodiesterase (PDE)
second messenger = DECREASE in GMP
Na+ channel closure

57. Photo- transduction in photoreceptor cells
In dark condition the rhodopsin are inactive cGMP-gated ion channels are opened the rod cell are depolarized high release of neurotransmitters
In light condition rhodopsin absorbs light and activates transducing cGMP decrease cGMP-gated ion channels become closed reduced the release of neurotransmitters.

58. Amplification of Photo- transduction
one rhodopsin activates 500s of G -GTP complexes, which in turn activate 500s of GMP phosphodiesterase , that each produce 105 of cAMP molecules, and so on.
The overall amplification associated with rhodopsin signaling result in the alteration of membrane potential of 1 mV

59. Stimulatory G-protein and Inhibitory G protein
Stimulatory G proteins (Gs) increase the cAMP concentration and activate PKA.
However several extracellular signals activate a different class of G proteins known as Inhibitory G proteins (Gi).
Inhibitors G protein blocks the Adenylyl Cyclase ( AC) activity thereby reducing cAMP levels.

60. Epinephrine-induced breakdown of glycogen.
Reception
Binding of epinephrine to G-protein-linked receptor (1 molecule)
Transduction
Inactive G protein
Best characterized example of Gs protein coupled receptor is the epinephrine receptor.
The binding of epinephrine to G-protein-linked receptor triggers a signaling pathway leading to the release of glucose from glycogen, which takes places in hepatocytes (liver cells).
Active G protein (102 molecules)
Inactive adenylyl cyclase
Active adenylyl cyclase (102)
ATP
Cyclic AMP (104)
Inactive protein kinase A
Active protein kinase A (104)
Inactive phosphorylase kinase
Active phosphorylase kinase (105)
Inactive glycogen phosphorylase
Active glycogen phosphorylase (106)
Breakdown of glycogen
to glucose monomers by
phosphorolysis in liver
and muscle cells
Response
Glycogen
Glucose-1-phosphate
(108 molecules)

61. Stimulatory G-protein and Inhibitory G protein
Both Stimulatory G proteins (Gs) and Inhibitory G proteins (Gi) are targets for bacterial toxins:
Cholera toxin stimulates Gs:
catalyzes ADP ribosylation of the α subunit of Gs and blocks its GTPase activity thereby compromising the GTP hydrolysis.
 As a result, α subunit of Gs becomes permanently “active” causing a persistent stimulation of adenylate cyclase and elevated cAMP  large efflux of water and Cl- in the gut that causes diarrhea.
Stimulatory G-protein and Inhibitory G protein

62. Stimulatory G-protein and Inhibitory G protein
Pertussin toxin stimulates Gi:
catalyzes ADP ribosylation of the α subunit of Gi keeping it in the inactive GDP bound state.

63. In class presentation on FeBRUARY 11, 2019
Establish 4 team groups (12 max people per group ): Each student should join a group of choice. (Each of you is free to join a group of interest but, if you prefer, I can make up the group)
Each team needs to:
Choose 1 out 4 scientific articles I have sent for today class;
Read the chosen article and make a 20 min presentation to be delivered for in class presentation on February 11;
The presentation should be shared among team members;
Be prepared to answer in class questions from the professor and your peers.
Please send to me temple.edu) the following:
Name of the students for each group and the paper chosen by February 7
PPT presentation along with information for each group by 12 AM on February 11.