SIGNAL TRANSDUCTION OVERVIEW Fahareen-Binta-Mosharraf MIC 404 1

Signal transduction describes the process by which a receptor interacts with a ligand at the surface of the cell and then transmits a signal to trigger a pathway within the cell. Two fundamental types of response 1.Material 2.Signal 2

1.Material—molecular or macromolecular—is physically transmitted from the outside of the membrane to the inside by transport through a proteinaceous channel in the lipid bilayer. 2. A signal is transmitted by means of a change in the properties of a membrane protein that activates its cytosolic domain 3

Physical transfer of material/DIRECT Channels control the passage of ions: 1.Different channels exist for potassium, sodium, and calcium ions 2.By opening and closing in response to appropriate signals Carrier transportation: 1.Transporters are responsible for the import of small molecules sugars across the membrane 2.The target molecule binds to the receptor on the extracellular side, but then is released on the cytoplasmic side Internalization/Endocytosis: 1.receptor-ligand combination is brought into the cell by the process of endocytosis 2.receptor and ligand are separated; the receptor may be returned to the surface for another cycle, or may be degraded. 4

Three membranes of transferring material 5

The transmission of a signal/Indirect Involves the interaction of an extracellular ligand with a transmembrane protein that has domains on both sides of the membrane. Binding of ligand converts the receptor from an inactive to an active form. 6

Basic principle of cell signaling 7

Classifications of Signal Transducing Receptors Receptors that penetrate the plasma membrane and have intrinsic enzymatic activity. Receptors that have intrinsic enzymatic activities include those that are tyrosine kinases (e.g. PDGF, insulin, EGF and FGF receptors) Receptors that are coupled, inside the cell, to GTP-binding and hydrolyzing proteins (termed G-proteins). Examples of this class are the adrenergic receptors, odorant receptors, and certain hormone receptors (e.g. glucagon, angiotensin, vasopressin and bradykinin). 8

Receptors having intrinsic enzymatic activity 9

Receptors that are coupled inside the cell with specific protein 10

Different kinds of intracellular signaling proteins along a signaling pathway from a cell-surface receptor to the nucleus. In this example, a series of signaling proteins and small intracellular mediators relay the extracellular signal into the cell, causing a change in gene expression. Two ways 1.Receptor transmits the signal to nearby enzymes called EFFECTOR which generates a SECOND MESSENGER (GPCR) 2.Receptor transmits the signal into a recruitment station for cell signalling proteins (RTKs)

Ultimately, the signaling pathway activates (or inactivates) target proteins that alter cell behavior. In this example, the target is a gene regulatory protein. Ultimate effect of signal transduction- 1.Transcription 2.Protein synthesis 3.Metabolic change 4.Movement 5.Survival 6.Cell death 12

Two types of intracellular signaling proteins that act as molecular switches. In both cases, a signaling protein is activated by the addition of a phosphate group and inactivated by the removal of the phosphate. (A) The phosphate is added covalently to the signaling protein by a protein kinase. (B) A signaling protein is induced to exchange its bound GDP for GTP.

14

15

Signaling by GTP binding/by heterotrimeric G protein 16

Heterotrimeric G protein G protein activates or inhibits target protein A G-protein is converted into active form when it interacts with an activated receptor. Receptor activates when its bound GDP is replaced by GTP G protein has three subunits-G α,G β and G γ G protein along with its activated receptor is called G protein coupled receptor (GPCR) Exist in two states 1) bound GTP: active 2) bound GDP: inactive

Why do we care about the structure of the structure of G proteins: including the G  and G  interface? The  subunit binds and hydrolyzes GTP GTP-  : dissociates from G  (tightly associated) Both subunits (  and , then activate their respective effectors). Following hydrolysis of GTP to GDP, subunits reassemble and become inactive Fig

The regulation of G proteins. 1. Initially G  has bound GDP, and  &  subunits are complexed together. 2. ligand binding, usually to an extracellular domain of a receptor (GPCR), causes a conformational change in the receptor that is transmitted to a G-protein on the cytosolic side of the membrane. Adenylate Cyclase

3.G  releases GDP & binds GTP (GDP- GTP exchange). 4. G  -GTP dissociates from the inhibitory  complex & can now bind to and activate an effector,eg-Adenylate Cyclase 5. Adenylate Cyclase, activated by the stimulatory G  - GTP, catalyzes synthesis of a second messenger,eg- cAMP 21

Turn off of the signal: 1. GTPase activity of G  hydrolyzes GTP to GDP The presence of GDP on G  causes it to rebind to the inhibitory  complex. Adenylate Cyclase is no longer activated.

2. Receptor desensitization varies with the hormone. In some cases the activated receptor is phosphorylated via a G-protein Receptor Kinase. The phosphorylated receptor then may bind to a protein  -arrestin.  -Arrestin promotes removal of the receptor from the membrane by endocytosis.

G-proteins are tightly regulated 3 types of accessory proteins that modulate cycling of G-proteins between GTP/GDP 1. GAPs : (GTPase-activating proteins). Stimulate GTP hydrolysis. Inactivate G-protein.

2. GEFs : (Guanine nucleotide-exchange factors) Stimulate dissociation of GDP (inactive) from G-protein so GTP can bind (active). 3. GDIs : (Guanine nucleotide-dissociation inhibitors) Inhibit release of bound GDP (maintain G-protein in inactive state). 26

Amplification meant signal transduction glucagon and epinephrine leads to glucose metabolism The concentration level of these hormones are very low(<10 -8 ) So binding of single hormone to specific receptor amplifies the signal system to activate large number of second messenger-cAMP in a short period of time. Thus production of high level second messenger is one of the most important step in glucose metabolism cascade referred to as amplification meant signal transduction. 27

Activation of cAMP and Protein Kinase A also play major roles in response of liver to glucagon or epinephrine Figure 15.7

Regulation of blood glucose level/Switch on of catabolic pathway 29

CREB pathway/Switching on Anabolic Pathway/Response on nucleus and its gene to cAMP Thus epinephrine and glucagon not only activate catabolic enzymes in glycogen breakdown they leads to the synthesis of anabolic enzymes required to form glucose smaller precursors 30

31

Receptor tyrosine kinase (RTKs) –Most prominent receptor with kinase activity. –Integral membrane protein. –Contain extracellular ligand binding domain. –Phosphorylates target protein on tyrosine residue. –>50 known RTKs are available 32

Activation of RTKs Two mechanisms for receptor dimerization: –ligand mediated –receptor mediated 33

ligand mediated In non- activated state receptors those are present in membrane are monomers. bivalent ligand have two binding sites so can bind with two receptors at a time and initiate dimerization. Eg; PDGF 34

receptor mediated In this case ligand is monovalent and separate ligand molecules binds to each of the inactivate monomeric receptors. 35

Consequence of dimerization dimerization of receptors causing a conformational change in cyoplasmic domain that activates tyrosine kinases catalytic activity. Results of a trans-autophosphorylation mechanism in which kinase activity of one receptor phosphorylates the tyrosine residue of other receptor of the dimer and vice versa. The newly formed phosphor-tyrosine residues of the receptors serve as binding sites for target proteins containing either SH2 or PTB domains and activate target proteins. 36

Phosphorylated tyrosine (RTK) served as docking sites for Specific domain containing protein Domains : Src homology 2 (SH2) domain The SH2 (Src Homology 2) domain is a structurally conserved protein domain present in many intracellular signal-transducing proteins also encoded by genes of tumor encoded (oncogenic) viruses Phospho-tyrosine binding (PTB) domain SH3 domain 37

Ras Pathway Ras is originally discovered as viral oncogene,that is carried by certain tumor viruses capable of transforming a normal cell to malignant state Characteristics Small monomeric G protein. Ras cycles between inactive GDP-bound form to active GTP bound form.like other G protein Ras is 300 a.a long similar to Gα protein. It is not linked to receptor tyrosine kinase. Two proteins are responsoible for activation of Ras- Grb2 (adaptor protein) and Sos (Sons of sevenless) 38

Steps Binding of GF such as EGF or PDGF causes dimerization and autophosphorylation of RTKs. The newly formed phosphotyrosine residues serve as binding sites for SH2 domain containing adaptor proteins Grb2 and Sos. These protein couple induce exchange of GTP for GDP and activate ras. The GTP activated Ras leads to activation of Raf (ser/thr kinase) which in turn triggers another cascade eg MAP kinase cascade 39

MAP(mitogen activated protein) Kinase cascade At least three different MAP kinase families and they provide important switching points in their pathways. They are activated in response to a wide variety of stimuli- 1.Stimulation of cell growth 2.Cell differenciation 3.Play a central role in controlling changes in cell phenotype 40

Steps of MAP kinase cascade 1.Activated Ras recruits the protein Raf to the membrane activating its ser protein kinase function. 2.Raf is also calles MAPKKK because it phosphorylates MAPKK (MEK). 3.MEK is a dual specificity kinase as it phosphorylates- Tyrosine as well as Threonine serine residue. 41

4. MEK phosphorylates MAPK (ERK) 5. Once activated MAPK it phosphorylates transcription factor (TF). 6. phosphorylation of TF increases their affinity for regulatory sites on DNA leading to an increase of transcription of specific genes involved in growth regulation 42

Ultimate Effect of MAP Kinase pathway 1. MAP kinase controls TF in three ways- MAP kinase directly translocates to nucleus and phosphorylates TF there. MAP kinase phosphorylates TF in cytoplasm, activated cytoplasmic function and then move to nucleus. MAP kinase phosphorylates a cyrtoplasmic inhibitor protein and causes release of TF. TF then move to nucleus. 43

2. Ativate trascriptional regulator myc,fos and jun 3. Inhibit Rb and release E2f-Dp1 45

46

Regulation of MAP cascade/Turn off MAP cascade When cell proliferation signal is off,TF stimulates another gene that codes for MAPK phosphatase (MKP-1) MKP-1 removes phosphate groups from MAPK thereby inactivating the kinase function of MAPK and preventing further cell proliferation signals. 47

48

Cross Talking Effector Phospholipase C is an effector can react in both types of receptors GPCR (with ligand hormones and neurotransmittesrs) and RTKs (with ligand GF) PLC activation by GPCR or RTKs it hydrolyzes and releases PIP2 (phosphatidyl inositol-4,5-bis phosphate) PIP2 releases second messenger DAG (diacetyl glycerol)and IP3 (inositol tri phosphate) 49

50

DAG activates in prsence of Ca 2+ activates PKC PKC activates ERK and Mdm2 ERK phosphorylate TF (NFκB) and Mdm2 block p53-both stimulate for cell proliferation 51

52

53

Calcium Ions as Second Messengers Calcium ions (Ca2+) are even more widely used than cAMP as second messengers in signal transduction pathways Increasing the cytosolic calcium concentration causes many responses in animal cells, including: 1. Muscle cell contraction 2.Secretion of certain substances 3.Cell division Cells use calcium ions as a second messenger in both G-protein and receptor tyrosine kinase 54

55