Cell surface receptors

ion-channel-linked

guanylylcyclases

tyrosinekinases

tyrosine kinaseassociated

tyrosinephosphatases

serin/threoninekinases

enzyme-linked G-protein-linked

Cinases

On the basis of amino acid :

Tyrosine kinases,

Serine threonine (PKC, Plk,Rho Kinases)

Receptor (EGFR,FGFR,PDGFR)

Non receptor (JAK,src,Abl,MAPK)

Receptor tirosina quinase Signal molecule

Signal-binding site

CYTOPLASM

Tyrosines

Signal molecule αHelix in the

Membrane

Tyr

Tyr Tyr

Tyr Tyr

Tyr Tyr

Tyr Tyr

Tyr Tyr

Tyr

Tyr

Tyr Tyr

Tyr Tyr

Tyr Tyr

Tyr Tyr

Tyr Tyr

Tyr

Tyr

Tyr Tyr

Tyr Tyr

Tyr

Dimer Receptor tyrosine kinase proteins (inactive monomers)

P P P

P P

P Tyr

Tyr Tyr

Tyr Tyr

Tyr P

P P

P P

P Cellular response 1

Inactive relay proteins

Activated relay proteins

Cellular response 2

Activated tyrosine- kinase regions (unphosphorylated dimer)

Fully activated receptor tyrosine-kinase (phosphorylated dimer)

6 ATP 6 ADP

The cytoplasmic domain is a protein kinase that phosphorylates Tyr residues (a Tyr kinase) in specific target proteins. The receptors for insulin and epidermal growth factor are prototypes for the approximately 60 RTKs in humans.

Growth factor receptors that initiate signals through Tyr kinase activity include those for insulin (INSR), vascular epidermal growth factor (VEGFR), platelet-derived growth factor (PDGFR), epidermal growth factor (EGFR), high-affinity nerve growth factor (TrkA), and fibroblast growth factor (FGFR).

Domain organization in a variety of receptor tyrosine kinase (RTK)

Some Signaling Proteins That Act Via Receptor Tyrosine Kinases

SIGNALING LIGAND RECEPTORS SOME RESPONSES

Epidermal growth factor (EGF) EGF receptor stimulates proliferation of various cell types Insulin insulin receptor stimulates carbohydrate utilization and protein synthesis

Insulin-like growth factors IGF receptor-1 stimulate cell growth and survival

(IGF-1 and IGF-2)

Nerve growth factor (NGF) Trk A stimulates survival and growth of some neurons

Platelet-derived growth factors PDGF receptors stimulate survival, growth, and proliferation of various cell types

Macrophage-colony-stimulating M-CSF receptor stimulates monocyte/macrophage

factor (M-CSF) proliferation and differentiation

Fibroblast growth factors FGF receptors stimulate proliferation of various cell (FGF-(FGF1 to FGF-24) (FGF-R1–FGFR4) types; inhibit differentiation of some precursor cells; inductive signals in development

Vascular endothelial growth VEGF receptor stimulates angiogenesis

factor (VEGF)

Ephrins (A and B types) Eph receptors (A and B) stimulate angiogenesis; guide cell and axon migration

Many signaling processes are mediated by an assembly of multiple signaling molecules.

Assembly of signaling molecules

Intracellular Signaling Pathways

Signal Termination

Receptor enzimático: A ligação do ligante ao domínio extracelular estimula a atividade enzimática no domínio intracelular.

O Receptor de Insulina é uma tirosina quinase que se auto-fosforila e em seguida cataliza a fosforilação de outras proteínas alvo.

Insulin regulates both metabolic enzymes and

gene expression

Insulin signal transduction.

Regulation of gene expression by insulin through a MAP kinase cascade

Insulin receptor: INS-R INS-R autophosphorylation Insulin receptor substrate -1 (IRS-1) Ras Raf-1 MEK: MAPKK ERK: MAPK Elk1 Serum response factor (SRF)

Cross talk between GPCR and RTK

Insulin - INS-R Phosphorylates β-adrenergic receptor. PKB Internalization of adrenergic receptor. Alternatively, INS-R–catalyzed phosphorylation of a GPCR INS-R uses GPCR to enhance its own signaling.

Receptor tyrosine kinase Growth factors: EGF, PDGF etc

Growth factors: EGF, PDGF

Mutant ras can bind GTP but can not hydrolyze it, and thus remain constitutively in “on” state Most oncogenic ras proteins contain a mutation in codon 12 (Gly). This blocks the binding of GAP to ras, and prevents GTP hydrolysis.

~30% of all cancers involve Ras mutations.

RTS signaling and Ras GTPase

RTK-RAS-MAPK cascade

RTK: kinase receptor Ras: GTPase MAPKKK: kinase MAPKK: kinase MAPK: kinase Transcription factors (e.g., c-Fos) Why multi-step in signaling? Signal amplified in number and duration Signal fine regulated Signal cross talks

The Ras-activated MAP kinase cascade.

The JAK-STAT transduction mechanism for the erythropoietin receptor

Erythropoietin (EPO) Dimerization of EPO receptor. JAK phosphorylates EPO-R. (a)STAT5 binds to P–EPO-R JAK - p-STAT STAT5 dimer Exposing a nuclear localization sequence (NLS). (b) Grb2 binds P–EPO-R and triggers the MAPK cascade.

Cytokines: paracrine/autocrine polypeptides Interleukins: immune response in leukocyte Interferons: immune response against viruses or bacteria

The JAK-STAT pathway for the intracellular relaying of cytokine

23

RTK = receptor tyrosine kinase

Shc = adapter protein tyr P by growth factors contains PTB, SH2 domains

and tyr P sites Grb2 = adapter protein with 1 SH2 & 2 SH3 domains

SOS = son of sevenless = GEF for Ras

Ras = small G-protein, part of p21 Ras family including H-Ras, K-Ras,

N-Ras, & R-Ras

cRAF1 = a tyrosine kinase

GAP = GTPase activating protein

MEKs = Map Kinase Kinases, unusual in that it will phosphorylate both

thr and tyr (at least 7) MEK = MAPK/ERK Kinase

ERKs = Extracellular signal Regulated protein Kinase; MAP kinases (mitogen activated kinases) phosphorylated on TEY motif (others include JNKs, SAPKs, & p38 kinase)

GEF = Guanine Nucleotide Exchange Factor Raf

MEK1/2

ERK1/2

Rsk , MSK1,

etc

Mitogenesis, Differentiation,

Proliferation, development, neuronal survival,

Memory formation

Transcription Factors

Cytoskeletal proteins

SOS

Shc Grb2

Ras

Hormone

Classic “Map Kinase” Pathway

MAP kinase cascades in mammalian cells.

Tyrosine kinase-derived oncogenes Oncogene Retrovirus cellular counterpart

v-src Rous sarcoma c-src v-erbB avian erythroblastosis EGF receptor v-fms McDonough feline sarcoma CSF-1 receptor v-kit feline sarcoma SCF receptor v-abl Abelson murine leukemia c-abl

v-sis simian sarcoma PDGF

Tyrosine kinase oncogenes are formed as a result of mutations that induce constitutive kinase activity.. Activated tyrosine kinase oncogenes generally cause enhanced proliferation and prolonged viability, but do not typically block differentiation. Common signaling pathways are involved in mediating these effects, including activation of phosphotidylinositol 3-kinases (PI3K), the Ras/Raf/MAP kinases, phospholipase C-γ (PLCγ), and Signal transducers and activators of transcription (Stats).

Int J Med Sci. 2004; 1(2): 101–115.

Int J Med Sci. 2004; 1(2): 101–115.

Int J Med Sci. 2004; 1(2): 101–115.

VEGFR-3 mutations in hereditary lymphedema

ss

VEGF-R3

S641P

P1114L

Original definition included congenital, bilateral or unilateral, chronic hereditary edema in lower extremities, causing inhibition in receptor autophosphorylation

Indivíduos portadores de uma mutação VEGFR3 exibem edema congênito dos membros inferiores, geralmente bilateralmente e abaixo dos joelhos, às vezes associado a celulite, veias proeminentes, papilomatose e hidrocele.

Clin Genet. 2006 Oct;70(4):330-5.

VEGFR-3 mutations in hereditary lymphedema

Interação proteína – proteína (Two hybrid)

The yeast two-hybrid assay uses two plasmid constructs: - Bait plasmid, which is the protein of interest fused to a GAL4 binding domain, - Hunter plasmid, which is the potential binding partner fused to a GAL4 activation domain.

The two-hybrid system.

Pull-down assays

https://www.thermofisher.com

Hanahan and Weinberg (2000) Cell 100: 57.

Signal Transduction Cross-Talk Plays a major Role in Biology