Cell Biology interactive media video or interactive 1 Lecture 4
& 5: Cell biology 2014 (revised 29/1 -14)
Slide 2
Cell communication DifferentiateProliferate xx DieSecrete Move
Signal molecules/proteins 2 All diseases involve changes of normal
cells. In some cases, these changes may affect other cells of the
individual
Slide 3
Events during cell communication Regulated synthesis...... or
regulated release of a signaling molecule (Transport of signaling
molecule to target cells) Binding of the signaling molecule to a
specific receptor on/in a target cell Activation of a transduction
chain 1. 2. 3. 4. Target cell response 5. Termination of signal 6.
1. 2. Target cell 3. 4. 5. Producer cell 3 6. hormone" = to urge
on/impulse
Slide 4
Signaling receptor diversity The mammalian genome encodes for
thousands of signaling receptors - Many of these are targets for
drugs Tissue specific expression: Each individual animal cell
express only some of these receptors 4
Slide 5
Membrane permeability O Hydrophobic molecules Na + Charged
molecules Ions Amino acids Cl - Large uncharged polar molecules
Glucose 5 CortisolTestosteroneCholesterol O N O O O N CC
Slide 6
Localization of signaling receptors Receptor on plasma membrane
Receptor in cytosolReceptor in nucleus Hydrophilic molecule (and
proteins) Hydrophobic molecules Other compounds than the natural
ligand may interact with a receptor some are used as drugs (legal
& illegal) natural ligand = an endogenous receptor binding
molecule 6 hydrophobic lipohilic non-polar (often used as
synonyms)
Slide 7
Receptor agonists and antagonists Other compounds than the
natural ligand may bind a receptor Agonists: mimic completely, or
partially, the action of the endogenous ligand Antagonists: bind to
receptor without activating it block the action of the natural
ligand AdrenalinPhenylephrine (natural)(selective agonist) One of
the action of adrenalin is to cause a dry mouth in the
fight-or-flight reflex. Phenylephrine is used in many cold-relief
drugs to prevent excessive nasal mucous secretion 7
Slide 8
A B C D E Five modes of cell communication Neuron Bloodstream B
Paracrine C Autocrine D Endocrine E Neuronal/synaptic Signaling by
secreted ligands: Contact dependent signaling: Ligands on the cell
surface A 8 Surface receptor Intra-cellular receptors "crinis" =
secrete
Slide 9
Signaling cell Target cell Receptor/ligand Contact-dependent
signaling uses ligands and receptors that are plasma
membrane-bound: - Persistent signals (uni- or bidirectional) -
Directed toward neighboring cells A Contact-dependent signaling
9
Slide 10
A Distinct types of contact-dependent cell signaling Cell
surface receptors that mediate cell-to-cell adhesion (cadherins)
and cell-to-ECM interaction (integrins) are also involved in
signaling. Important for: Development Growth control Survival Gap
Junctions permit free passage of small molecules between adjacent
cells Important for e.g., synchronous heart contraction Cadherin
Gap junction Integrin 10
Slide 11
Paracrine signaling involves secretion of a ligand that act
locally on cells with the appropriate receptors: Local effect
"para" = near Signaling cell Adjacent target cells B Paracrine
signaling 11
Slide 12
Signaling and target cell Autocrine signaling implies that a
cell secretes a ligand that it responds to itself C Autocrine
signaling ? 12 "autos" = self
Slide 13
Endocrine signaling involves a signal molecule (poly-peptide or
steroid hormone) produced by an endocrine cell. "endo" =
inside/within "crinis" = secrete Each endocrine cell secrete only
one type of signal molecule! The hormone travels through the blood
system: Global signaling with long-term effect Relatively slow
responses - the signaling molecule have to travel through the blood
systems before reaching a target cell Endocrine cell secreting
Distant target cells D Endocrine signaling 13
Slide 14
Signaling cell "syn" = together "haptein" = hold onto
Neuronal/synaptic signaling is mediated by neurotransmitters
released at the interface between the signaling and the target
cell, called synapse. The release of neurotransmitters at the
synapse is controlled from the cell body through electrical
signals. Neurotransmitters bind cell surface receptors. - Acts
rapidly and transiently on the target cells Target cell Cell body
of a neuron Axon Synapse Release of neurotransmittor E
Neuronal/synaptic signaling 14
Slide 15
kidney *Gland Neuroendocrine integration Hormone secreting
glands in the brain link neuronal signals and peripheral endocrine
glands. Fight-or-flight reflex: the Hypothalamic- Pituitary-Adrenal
(HPA) system The adrenal gland responds to both the hormone (ACTH)
and a nerve signal ACTH Adrenal Cortex cortisol Increased blood
levels of lipids etc. etc Nerve signal adrenal medulla adrenaline
Increased blood levels of lipids & glucose etc. etc. Endocrine
cell: a cell within an endocrine gland that release a hormone into
the circulating blood in response to a neural (synaptic) or
hormonal stimulus 15 *Gland
Slide 16
Signaling molecules Molecules typically produced and released
by one cell and recognized by another cell Signaling molecules are
chemically diverse: - Gases: nitric oxide, carbon monoxide -
Steroids: testosterone, cortisol, etc. - Proteins: insulin,
glucagon, etc. - Amines: catecholamines, acetylcholine Ryss 5a: A
mix of synthetic anabolic steroids ( muscle growth) Membrane
permeable Membrane impermeable 16
Slide 17
Fast versus slow signal transduction events Altered protein
function Cell response An altered cytoplasmic signaling protein DNA
mRNA Altered gene expression Altered protein level mRNA Protein
Slow (minutes to hours) Fast (
I. Molecular switches in signal transduction A signal that can
be switched on, also needs to be switched off (all signals are more
or less transient) 1. Protein phosphorylation The most common
on-off switch is provided by protein phosphorylation OH Serine,
threonine or tyrosine Serine, threonine or tyrosine O O O - P O
Kinase - Phosphatase 26 + ATP Kinase : ~1000 protein kinase genes
in vertebrates. Some have only a single substrate. Others are
multi- functional and may have >10 substrates
Slide 27
GTP GDP Guanine-nucleotide Exchange Factor (GEF) GTPase
Activating Protein (GAP) Inactive Active P GDP II. Molecular
switches in signal transduction 2. GTP binding proteins
(G-proteins) Another on-off switch is provided by regulatable
GTP-binding and hydrolysis GDP GTP >> GTP 27 Molecular_models
15.5-Ras (one PO 4 makes the diff.)
Slide 28
Signal transduction cascades MetabolismGene regulation Etc. P
GTP A single cell surface receptor may activate several signal
transduction pathways This involves various G- proteins, 2nd
messengers and protein kinases Protein kinases at the end of a
cascade may have many substrates Response: 28 PP Kinase Ca 2+ cGMP
cAMP P. M.
Slide 29
Three main classes of cell-surface receptors G-protein coupled
receptors Receptors with intrinsic enzymatic activity ZZZ 29
Slide 30
3. G-proteins may regulate enzymes or ion channels G-protein
coupled receptors (GPCR) A hallmark of GPCRs is 7 trans- membrane
spanning regions ZZZ 1. Ligand binding conformational change 2. A
specific G-protein is recruited and activated G 30
Slide 31
Down-stream effectors of various G-proteins Cyclic AMP Adenylyl
cyclase ATP Guanylyl cyclase Cyclic GMP GTP Phospholipase C Ca 2+
Increase in cytosolic and activation of protein kinase C Ion 1.2.
3.4.Ion channels 31
Slide 32
I. Regulation of hetero-trimeric G-proteins GTP GDP Inactive
Active P GDP GTP + subunit and/or -subunit can activate or suppress
different downstream targets Complex dissociate upon GTP binding
=GAP GDP GTP >> 32 RGS =GEF RGS: Regulator of G-protein
Signaling
Slide 33
P.M. GDP GTP + GDP GTP Ligand binding causes a conformational
change The G-protein is recruited to the receptor, which acts as a
GEF the -subunit exchanges GDP for GTP dissociation of an active
-subunit GDP No ligand (default state) 33 II. Regulation of
hetero-trimeric G-proteins
Slide 34
GTP P.M. GDP P + The intrinsic GTP hydrolysis is slow but RGS,
an -subunit specific GAP, catalyzes hydrolysis. This terminates the
signal RGS GTP ss ii Phospholipase C- (PLC- ) GTP qq Adenylyl
cyclase A family of - subunits with distinct functions Anim.
15.3-G-protein_signaling 34 III. Regulation of hetero-trimeric
G-proteins Alberts et al: Table 15-3 ( tissue specificity )
Slide 35
Adenylyl cyclase activation by the s -subunit of G-proteins GTP
ss P.M. PPP PP P + Cyclic AMP ATP P AMP Adenylyl cyclase Cyclic-AMP
phosphodiesterase (constitutively active) Caffeine 35
Slide 36
Inactive PKA Cyclic AMP Active PKA Target genes CREB P Cyclic
AMP second messenger signaling Glycogen phosporylase P Glycogen
phosporylase Cyclic AMP activates Protein kinase A (PKA), which can
regulate: Metabolism Gene transcription 1. 2. 1. 2. Glucose-1-
phosphate Glycogen 36
Slide 37
Summary of the cyclic AMP signaling cascade Cyclic AMP CREB P
Adenylyl cyclase PKA Regulates metabolism ATP P Regulates
transcription Glycogen breakdown P GTP ss Anim. 15.4-cAMP_signaling
37 -Regulated DNA binding P Glycogen: -Stored in muscles and liver
-Rapidly available energy source Work/stress adrenalin cAMP PKA
Glycogen breakdown Alberts et al: Table 15-1 ( tissue specific
response ) GEF (GPCR)
Slide 38
Signal induced cleavage of phospholipids Variable Phosphate
Glycerol Fatty acid Phospholipase A 1 Phospholipase A 2
Phospholipase C Phospholipase D External signals may activate
distinct phospholipases that cleave phospholipids at specific sites
and thereby catalyze the formation of various molecules with
signaling properties Soluble compounds release into the cytosol 38
Precursors for various signaling substances
Slide 39
Phospholipase C activation generates two 2nd messengers qq
Fatty acid Glycerol Fatty acid Glycerol OH Diacylglycerol (DAG)
Phosphatidylinositol 4,5- bisphosphate, PI (4,5)P 2 Inner leaflet
of plasma membrane Phospho- lipase C- (PLC- ) P P P P P P Inositol
1,4,5- triphosphate, IP 3 q -subunit activates PLC 1. 2.PLC cleaves
PIP 2, generating the two 2nd messengers DAG and IP 3 1. 2. GTP
39
Slide 40
P P P IP 3 OH Inner leaflet of plasma membrane Ca 2+ IP 3
regulated Ca 2+ channel PKC Ca 2+ PKC Ca 2+ Calmodulin Ca 2+
Calmodulin Calmodulin regulated Ca 2+ pump in ER Ca 2+ DAG 1. 2.
DAG recruits PKC to plasma membrane IP 3 mediate release of Ca 2+
from ER 3.DAG and Ca 2+ activates PKC 1. 2. 3. 4.Ca 2+ activates
calmodulin to terminate signal by pumping Ca 2+ back into ER 4.
Role of the 2nd messengers IP 3 and DAG 40
Slide 41
Ca 2+ /calmodulin dependent protein kinase (CaMK) P Calmodulin
Ca 2+ Calmodulin Ca 2+ Calmodulin Ca 2+ Autophosphorylation
Dephosphorylation Catalytic Inhibitory Calmodulin Activated Fully
active P Partially active Resting state Inactive Ca 2+ Increased
cytosolic 41 Molecular_models 15.6-calmodulin
Slide 42
IP 3 OH Ca 2+ PKC Calmodulin Ca 2+ DAG PLC- P P P P P P CaMK
Summary of G-protein signaling through PLC- qq GTP Other regulated
enzymes Ca 2+ STOP Termination of signal Ca 2+ Etc! 42 Both PKC and
CaMK have many potential (tissue specific) substrates GEF
(GPCR)
Slide 43
Enzyme linked receptors Tyr P Ser/Thr Many variants on this
theme here we focus on: Receptor tyrosine kinases Receptor
serine/threonine kinases P Homo-dimers Hetero-dimers Single pass
transmembrane receptors. Ligand binding cause dimer formation and
consequent auto-phosphorylation 43 Jenkinson : RTK - dimerization
Alberts et al: Table 15-4 (tissue specific RTKs)
Slide 44
Signaling through Receptor Tyrosine Kinases Tyr Kinase domain
Tyr Kinase domain Tyr Kinase domain Tyr Kinase domain Inactive
receptor monomers Active receptor dimer Ligand binding causes
receptor dimerization PP P. M. Trans-phosphorylation of tyrosine
residues Single pass transmembrane protein Tyr PP PP 44 Cis- prefix
means "on this side" Trans- prefix means "across"
Slide 45
Tyr PP PP Kinase domain Tyr Kinase domain PP SH2 SH3 GTP Ras
Ras GEF (Sos) P. M. Regions containing phospho-Tyr may serve as
specific docking sites for SH2 domain-containing signaling proteins
(SH = Src Homology domain) SH2-proteins binds at specific
phospho-tyrosines P 3 These can be enzymes. Phosphatidyl- inositol
(PI) Monomeric G-protein GDP Ras PI-3 Kinase 45 .or they act as
adaptors for signaling proteins Fig. 15-55
Slide 46
Phosphorylation cascade downstream of Ras GTP Ras Mek Raf P Erk
(MAPK) P. M. Erk P Target genes P 2. Erk P P Cytosolic target
proteins 1. Altered protein function Altered gene expression 2.
46
Slide 47
Termination of RTK/Ras/MAPK pathway Receptor and ligand
internalization GDPGTP Ras Erk (MAPK) Erk P 1.2.Ras GTP hydrolysis
Ras GAP 3.Dephosphorylation Phosphatase Note: Signaling receptors
are rarely recycled 47 Anim. 13.3-receptor_endocytosis (Note:
vesicle fusion with endosome) Fusion with endosome Fusion with
primary lysosome degradation
Slide 48
3 4 5 P PI kinasePIP kinase PI(4,5)P 2 P PI phosphorylation
cycles on inositol ring position 4 & 5 P P P P PI(4)P Inositol
Extracellular space Cytosol Phosphatidylinositol (PI) I. PI-kinases
act at specific positions of the inositol ring Phosphate Glycerol
Fatty acid Inositol 48
Slide 49
II. PI-3 kinase completes a PH-domain binding site PI(3,4,5)P 3
P 33 PI(4,5)P 2 PTEN PI-3 Kinase Activated receptor recruits and
activates PI-3 kinase1. 2. PI-3 kinase PI-3 kinase phosphorylates
PI(4,5)P 2 to generate PI(3,4,5)P 3, which will serve as a
docking-site for a family of signaling proteins with a PH-domain
(PH= Pleckstrin Homology) 1. 2. 3. PTEN removes phosphorylation on
position 3 on PI(3,4,5)P 3 to terminate signal 3. P P P P P
Phosphatidyl- inositol (PI) 49 PP
Slide 50
III. PKB/Akt activation downstream of PI-3 kinase P P PDK1
PKB/Akt P 33 PH-domains PKB/Akt P P P 33 PDK1 PKB/Akt PPPP P PP P
PDK1 phosphorylates PKB/Akt thereby mediating its activation 2. 1.
PI(3,4,5)P 3 brings PDK1 and PKB/Akt into proximity through their
PH-domains 1. 2. 50 PPPP
Slide 51
IV. Different signaling pathways same target P PI-3 K P P 3 GDP
GTP + PI-3 K Both G-protein- and RTK signaling may result in
generation of PI(3,4,5)P 3 There are two distinct PI-3 kinases
which differ in their regulatory domains Thus, a PI-3 kinase may be
recruited to the plasma membrane via a -subunit binding domain or a
SH2 domain 51 P
Slide 52
I. Transcriptional regulation by TGF- / BMP P.M. Target genes
PP Smad 2/3 P Smad 4 Smad 2/3 P Smad 4 Type I receptor Smad 7
Negative feedback loop Type II receptor: Ser/Thr kinase 52
TGF-
54 Signal transducing proteins are often targets of therapeutic
drugs or infections agents My own favorite protein! "All science is
either physics or stamp collecting" Ernest Rutherford (1871-1937,
Nobelprize1908) Recommended reading Chapter 15 879-941 946-954
Alberts et al. 5 th edition
