Presentation submitted to RGTU-UTD (SOPS) on:-

SUBMITTED BY:

DEEKSHA BOKRE

M.PHARM(Q.A)

Ist SEM

TOPIC : Receptor Tyrosine Kinases (RTKs)

CONTENTS

Introduction to cell signaling

Types of Receptors

Receptor tyrosine kinases (RTK)s

Mechanism action of insulin

Activation of RTKs via Ligand Binding

Recruitment of Signal Transduction Proteins to Activated Receptors

RTKs and Ras/MAP Kinase Signaling

RTK Activation of Ras

Ras Activation of MAP Kinase

MAP kinase cascade

Relationship between Signaling Pathways

Introduction to cell signaling

• Many biological processes require various cells to work together and to coordinate their activities. To make this possible, cells have to communicate with each other, which is accomplished by a process called cell signaling. Cell signaling makes it possible for cells to respond in an appropriate manner to a specific environmental stimulus.

• Cell signaling is also intimately involved in the regulation of cell growth and division. This makes the study of cell signaling crucially important for understanding how a cell can lose the ability to control cell division and develop into a malignant tumor.

Depending on the distance that the signaling molecule has to travel, we can talk about three types of signaling:-

endocrine signalingparacrine signalingautocrine signaling

Types of Receptors• There are a number of receptor classes that are

used in different signaling pathways.

• Protein-tyrosine kinases are enzymes that phosphorylate specific tyrosine residues on protein substrates.

• Protein-tyrosine phosphorylation is a mechanism for signal transduction that appeared with the evolution of multicellular organisms.

• Over 90 different protein-tyrosine kinases are encoded by the human genome. These kinases are involved in the regulation of growth, division, differentiation, survival, attachment to the extracellular matrix, and migration of cells.

Protein-tyrosine kinases can be divided in two groups:

Receptor protein-tyrosine kinases

Non-receptor protein-tyrosine kinases

They activated directly by extracellular growth and differentiation factors such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) or by metabolic regulators such as insulin

They are regulated indirectly by extracellular signals and they control processes as diverse as the immune response, cell adhesion, and neuronal cell migration.

• Receptor tyrosine kinases (RTK)s are the high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones.

•  Receptor tyrosine kinases have been shown not only to be key regulators of normal cellular processes but also to have a critical role in the development and progression of many types of cancer.

Receptor tyrosine kinases (RTK)s

• RTK’s need to form dimer in plasma membrane.

• It add phosphate group to specific tyrosine residue of other protein.

• It constitute of a super family distinct from the serine and theronine kinases that operate in the reaction cascade.

• It regulate cell growth and cell differentiation rather than the control of intermediary metabolism because insulin receptor has this enzymatic activity, it is referred to as RTK.

• RTK’s do not phosphorylate every tyrosine in substrate protein;they phosphorylate only those that are present within certain amino seqences called as phosphotyrosine motifs.

• The ligands for RTKs are soluble or membrane-bound peptide/protein hormones including nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and insulin.

Mechanism action of insulin

• The insulin receptor is a member of the tyrosine kinase superfamily, generally involved in cell growth and differentiation.

• The insulin receptor tyrosine kinase (RTK) is a tetramer of two extracellular α subunits and two transmembrane β subunits with a single transmembrane region.

• α subunits bind insulin and induce a conformational change in the β subunits, activating their cytoplasmic kinase domain.

• Activated β subunits phosphorylate one another and a variety of insulin receptor substrates (IRSs).

• RTKs phosphorylate only phosphotyrosine motifs ,After phosphorylation these motifs have a high affinity for SH2 domains.

• Interaction of phosphotyrosine motifs with SH2 proteins cause their conformational change so that they bind to other proteins;faciliate transmission of signals

Insulin stimulation activates Protein Kinase B and MAP kinase

• Most RTKs are monomers that only dimerize when bound to their ligand. Dimerization activates the kinase activity and leads to autophosphorylation, creating sites for interactions with specific effectors. A key component of the RTK cascade is Ras.

• Ras: It is a small g protein that is held at the inner surface of the plasma

membrane by a lipid group, Ras also acts as both a switch and a molecular timer. Ras consists of

only a single small subunit. In its active form Ras stimulates effectors in signalling pathway.Ras

has a very weak GPTase activity. Mutation in Ras-GAP gene causes neurofibromatosis 1.

• Ras proteins are present in two different forms: an active GTP-bound form and an inactive GDP-bound form.

• Ras-GTP binds and activates downstream signaling proteins. Ras is turned off by hydrolysis of its bound GTP to GDP.

• These proteins are involved in the regulation of numerous processes, including cell division, differentiation, gene expression, cytoskeletal organization, vesicle trafficking, and nucleocytoplasmic transport.

• Ras is mutated in 30% of human tumors. Most mutations block hydrolysis keeping ras active and the cell in a proliferative state.

• Grb2(growth factor receptor bound protein) It is an adaptor protein involved in signal communication.

In human it is encoded by GRB2 gene.

• Sos It is a guanine nucleotide exchange factor for Ras.

• Raf

Ras-GTP recruits raf, which becomes activated as a protein kinase and initiates the MAP kinase cascade

The binding of ligand activates the kinase domains which cross-phosphorylate the two monomers of the dimeric receptor. Phosphorylation first occurs at a regulatory site known as the activation lip.

Phosphorylation of the lip causes conformational changes that allow the kinase domain to phosphorylate other tyrosine residues in the receptor and in signal transduction proteins.

Activation of RTKs via Ligand Binding

Signal transduction system proteins interact with phosphorylated RTKs via phosphotyrosine binding domains(PTB) and SH2 (Src homology domain-2) within signal transduction proteins such as the multi-docking protein known as the insulin receptor substrate-1 (IRS-1) perform this function

The binding of signaling proteins either directly to the receptor or to IRS-1 allows them to be phosphorylated by the receptor. Some of these signaling proteins are involved in activation of the Ras GTPase .

Some such as phosphatidylinositol-3 kinase (PI-3 kinase) participate in lipid-mediated signaling pathways.

Recruitment of Signal Transduction Proteins to Activated Receptors

• Nearly all RTKs signal via Ras/MAP kinase pathways.

• They also may signal via other pathways. For example, the insulin receptor uses the Ras/MAP kinase pathway to regulate gene expression and the PI-3 kinase pathway to regulate enzyme activity (e.g., glycogen synthase).

• RTK-Ras/MAP kinase signaling controls cell division, differentiation, and metabolism.

RTKs and Ras/MAP Kinase Signaling

Ras once activated,it propagates signaling further inside the cell via a kinase cascade that culminates in the activation of members of the MAP kinase family.

MAP kinases phosphorylate TFs that regulate genes involved in the cell cycle and in differentiation.

Mutant RTKs or Ras/MAP kinase signaling proteins are associated with nearly all cancers.

Dominant Ras mutations that block GAP binding and lock Ras in the "on" state promote cancer.

RTK Activation of Ras

Step 1

EGF binding causes receptor dimerization and autophosphorylation on cytosolic tyrosines.

Step 2

The adaptor protein GRB2 binds receptor phosphotyrosine residues via its SH2 domain. GRB2 contains SH3 domains that allow the GEF protein known as Sos to bind to the membrane complex. Sos then recruits Ras to the complex. In the last step of Ras activation

Step 3

Sos promotes GTP exchange for GDP on Ras. The activated Ras-GTP complex then dissociates from Sos. The active form of Ras then activates the MAP kinase portion of the signaling pathway

Ras Activation of MAP Kinase

• Ras activates MAP kinase via a phosphorylation cascade that proceeds from Ras to Raf kinase, to MEK kinase, and finally to MAP kinase . MAP kinase then dimerizes and enters the nucleus

MAP kinase cascade(Mitogen-Activated Protein: activated by a mitosis-

stimulating growth factor)

Relationship between Signaling Pathways

G protein-coupled receptors, RTKs and integrins can all relay signals that result in the recruitment to the membrane of the adaptor protein Grb2 and the activation of Ras and the MAP kinase cascade.

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