Week 2 L3 - Kinase-Linked Receptors

Class 3 Receptors

Kinase-linked receptors

• where phosphorylation serves as a key regulatory mechanism

Examples of Kinase-linked receptors

Growth Factor receptors and insulin receptors.

What is auto-phosphorylation?

The process by which kinase-linked receptors phosphorylate themselves upon activation to trigger downstream signaling.

What are SH2 domains?

Specific protein domains that bind to phosphorylated Tyrosines (or Threonines and Serines) on the receptor.

what is the Kinase Cascade

A series of sequential phosphorylation events and binding of other protein domains triggered after SH2 domain binding.

What are the Downstream Target Proteins of Kinase Cascades

Enzymes, receptors, ion channels, transporters, transcription factors, contractile proteins, and secretory mechanisms.

Primary Cellular Functions of Class 3 Receptors

They are involved in cell growth and differentiation, and act indirectly by regulating gene transcription.

Name some responses as a result of kinase linked receptors being activated

• Physiological responses

• Immune responses

• Apoptosis

• Malignant transformation

• Growth

• Differentiation

Sequence of events following ligand binding to a Class 3 receptor

Ligand binding → Auto-phosphorylation → SH2 domain binding → Kinase cascade activation → Target protein modulation.

Pathological outcome of dysregulated Kinase-linked receptor signaling

Malignant transformation (cancer/uncontrolled cell proliferation).

explain what is happening in this image

• insulin binds to alpha subunits which are outside the cell

• flips a molecular switch, causing beta subunits to auto-phosphorylate

• Tyrosines are phosphorylated and exposed

• intracellular signalling protein (PI3K) docks onto them via SH2 domain

• signalling cascade is triggered

• PI3K signals GLUT vesicles that hold glucose transporters (GLUT) to move and fuse with cell membrane

• puts glucose transporters on the surface of the cell, allowing glucose to enter the cell.

what is the Baseline state of Receptor Tyrosine Kinases (RTKs) in the absence of a ligand

They exist as inactive, unphosphorylated monomers floating separately in the plasma membrane.

• Each monomer consists of an extracellular (EC) domain, a transmembrane (TM) segment, and an intracellular kinase domain.

First structural consequence of ligand binding to the extracellular (EC) domain

(step 3 in RTK structure and activation mechanism)

It induces receptor dimerisation (two separate monomer receptors physically pair up to form a dimer).

what happens in step 2 of RTK activation

A specific signaling molecule (ligand) binds to the extracellular domain of the receptor monomers.

what happens in step 3 of RTK activation

Ligand binding forces two adjacent receptor monomers to physically come together (dimerisation).

This close contact triggers trans-autophosphorylation, where the intracellular kinase domains cross-phosphorylate each other's tyrosine residues using ATP.

what happens in step 4 of RTK activation

• The newly formed phosphotyrosines on the intracellular tail serve as specific high-affinity docking sites.

• Intracellular SH2-domain proteins (such as Grb2, PI3K) recognize these sites and physically bind to the receptor, initiating downstream signaling cascades.

Overview of RTK structure and activation mechanism

• ligand binding induces receptor dimerisation

• triggers trans-autophosphorylation of tyrosine residues

• phosphotyrosines serve as docking sites for SH2 domain proteins (e.g Grb2, PI3K)

• initiates downstream signalling cascade

What happens once the SH2 domain (from Grb2) binds to the phosphorylated RTK

It recruits and triggers Activation of Ras via GDP/GTP exchange (Ras releases inactive GDP and binds active GTP).

What is the first kinase activated directly downstream of active Ras-GTP

Raf

What is the protein phosphorylated and activated directly by active Raf

Mek

What is the downstream target phosphorylated and activated directly by active Mek

MAP kinase (Mitogen-Activated Protein Kinase, also known as ERK).

What is the cellular destination of MAP kinase once it is phosphorylated

It translocates into the nucleus to phosphorylate various target proteins.

What are the direct molecular targets of MAP kinase inside the nucleus

Various transcription factors that bind to DNA.

Ultimate cellular outcome of the Ras/MAPK kinase cascade

Regulation of gene transcription to drive processes like cell growth, proliferation, and survival.

What is the Ras-MAPK pathway

A highly conserved intracellular signaling cascade where an activated Ras G-protein sequentially activates a series of kinases (Raf → Mek → MAP Kinase) to transmit signals from the cell surface to the nucleus.

Overview of the Ras-MAPK pathway

• Ras

• Raf

• Mek

• MAP kinase

• various transcription factors

• gene transcription (cell proliferation/differentiation)

What are the key ligands for the EGFR / ErbB receptor family

• EGF (Epidermal Growth Factor)

• TGF-alpha (Transforming Growth Factor-alpha).

Physiological roles of EGFR / ErbB signaling

Regulation of cell proliferation and differentiation.

Clinical relevance and pathology of dysregulated EGFR / ErbB

It is overexpressed in many cancers, driving uncontrolled cell division and tumor growth.

Key ligands for the Insulin R / IGF-1R family

Insulin and IGF-1 (Insulin-like Growth Factor 1).

Primary physiological roles of the Insulin R / IGF-1R family

Regulation of metabolism (e.g., glucose uptake via GLUT translocation) and cell survival.

Major clinical conditions linked to Insulin R / IGF-1R dysfunction

Diabetes (metabolic dysfunction/resistance) and cancer (via over-activation of survival pathways).

what are the only amino acids that can be phosphorylated

• tyrosine

• threonine

• serine

what specific enzymatic classification is given to PI3K upon its recruitment by an activated RTK?

lipid kinase

• primary function is to phosphorylate membrane phospholipids rather than protein residues

Which specific membrane lipid conversion is directly catalyzed by an activated PI3K?

PIP₂ to PIP₃

In the PI3K/Akt pathway, what is the effect of Akt on the protein BAD and its physiological outcome

Akt inhibits BAD, which acts as a pro-survival mechanism (blocking apoptosis/cell death).

In the PI3K/Akt pathway, what is the target and metabolic function of the mTOR pathway downstream of Akt

Activated mTOR drives protein synthesis and overall cell growth.

In the PI3K/Akt pathway, what is the effect of the pathway on GSK-3beta and its metabolic role

inhibits GSK-3beta, which plays a regulatory role in glycogen metabolism.

Three primary cellular outcomes of the PI3K / Akt Pathway

1. Cell survival

2. Protein synthesis

3. Metabolism

What is the PI3K/Akt pathway?

• activated RTK

• PI3K (lipid kinase)

• phosphorylates PIP₂ to PIP₃

• activates Akt (PKB)

• inhibits BAD (pro-survival) and activates mTOR

• mTOR inhibits GSK-beta (glycogen metabolism), and directly stimulates protein synthesis and translation machinery.

  • cell survival, protein synthesis, metabolism

What mutations result in most cancers

mutations of Ras (30% cancers) and PI3K pathway are most common oncogenic drivers

which pathways are major targets for anti-cancer drugs

Ras and PI3K pathways

• MEK inhibitor - trametinib

• mTOR inhibitor - everolimus

• PI3K inhibitor - alpelisib

What is the JAK/STAT Pathway (Cytokine Receptor Signaling)

A direct signaling pathway used by cytokines and growth hormones. - -

• Unlike RTKs, the receptors do not have built-in kinase activity; they must recruit separate JAK proteins to phosphorylate the receptor and activate STAT transcription factors.

Key components of the JAK/STAT pathway

• Cytokines: Interferons (IFN-alpha, IFN-gamma), Interleukins (IL-2, IL-6), Erythropoietin (EPO), and Growth Hormone (GH).

• JAKs (Janus Kinases): Soluble tyrosine kinases constitutively bound to the receptor tails. JAK1, JAK2, JAK3, TYK2

• STATs: Latent cytoplasmic transcription factors containing SH2 domains STAT1-6

Clinical & Pharmacological Relevance of JAK/STAT pathway

JAK Inhibitors (e.g., Tofacitinib, Baricitinib, Ruxolitinib):

• Used to treat autoimmune and inflammatory conditions like rheumatoid arthritis, myelofibrosis, and atopic dermatitis by blocking multiple cytokine signals simultaneously.

Key structural distinction between Cytokine Receptors and RTKs

Cytokine receptors lack intrinsic kinase activity; they cannot phosphorylate themselves and must rely on non-covalently associated JAK proteins instead.

JAK definition and its relationship to the resting receptor

Janus kinase; these are tyrosine kinase proteins that are constitutively associated (permanently attached) to the intracellular tail of the receptor.

4 key functional classes of cytokines/hormones that signal via JAK/STAT

• Interferons (IFN-alpha, IFN-gamma) — Antiviral/immune regulation

• Interleukins (IL-6, IL-2) — Inflammation/immunity

• Erythropoietin (EPO) — Red blood cell production

• Growth hormone (GH) — Growth/metabolism

describe the JAK/STAT pathway

• ligand binds to receptor

• receptor dimerisation

• 2 adjacent JAK domains phosphorylate each other (auto)

• emergence of active binding site SH2 domain of STAT

• JAK activates and phosphorylates STAT

• STAT dimerisation

Where does the JAK/STAT get the tyrosine kinase benefit from

from JAK family

• JAK is coupled to the receptor

• rely on JAK proteins for signal transduction

• cytokine receptors lack intrinsic kinase activity

what does STAT standard for

signal transducer and activator of transcription

Pharmacological Targeting of Kinase Receptors

Target site distinction: Monoclonal Antibodies vs. Small-Molecule TKIs

• Monoclonal antibodies target the extracellular domain of the receptor.

• Small-molecule TKIs penetrate the cell to target the intracellular ATP-binding site.

Pharmacological Targeting of Kinase Receptors

General mechanism of action for therapeutic Monoclonal Antibodies (mAbs)

They physically block ligand binding to the extracellular domain and can trigger antibody-dependent cellular cytotoxicity (ADCC) to recruit immune-mediated killing of the target cell.

Pharmacological Targeting of Kinase Receptors

General mechanism of action for Small-Molecule TKIs

They directly compete with ATP for binding within the intracellular kinase domain, preventing receptor trans-autophosphorylation and halting downstream signaling cascades.

Pharmacological Targeting of Kinase Receptors

Administration routes: Monoclonal Antibodies vs. TKIs

mAbs are large proteins and must be administered via IV infusion. TKIs are small chemical molecules and can be taken orally.

Pharmacological Targeting of Kinase Receptors

Selectivity profile comparison: mAbs vs. TKIs

mAbs are highly specific for a single target. TKIs have variable selectivity and can be multi-targeted (inhibiting multiple kinases simultaneously).

Pharmacological Targeting of Kinase Receptors

High-yield examples of therapeutic Monoclonal Antibodies and their targets

• Trastuzumab: Targets HER2

• Cetuximab: Targets EGFR

• Bevacizumab: Targets VEGF

Pharmacological Targeting of Kinase Receptors

High-yield examples of Small-Molecule TKIs and their targets

• Imatinib: Targets BCR-ABL, PDGFR, and c-KIT

• Erlotinib / Gefitinib: Targets EGFR

• Sunitinib: Multi-target kinase inhibitor

Pharmacological Targeting of Kinase Receptors

why are mABs and small molecule TKIs compared

they are the two main therapeutic strategies we have for shutting down overactive kinase receptors in diseases like cancer.

Primary therapeutic target of Imatinib in Chronic Myeloid Leukaemia (CML)

The BCR-ABL fusion kinase (an abnormal, constitutively active tyrosine kinase created by the Philadelphia chromosome translocation).

Precise molecular binding mechanism of Imatinib

It binds specifically to the ATP pocket of the inactive kinase conformation, locking it in an inactive state.

Secondary targets of Imatinib and their clinical application

It inhibits c-KIT and PDGFR, making it highly effective in treating Gastrointestinal Stromal Tumours (GIST).

4 major mechanisms by which cancer cells develop resistance to kinase inhibitors

1. Secondary mutations in the kinase domain (e.g., EGFR T790M).

2. Gene amplification (producing more of the target receptor to overwhelm the drug).

3. Pathway bypass (activating a parallel pathway, e.g., MET amplification).

4. Downstream mutations (mutations in downstream effectors like Ras or BRAF that keep the pathway on even if the receptor is blocked).

General methods used to shut down EGFR signaling in cancer therapy

Extracellular blockade using monoclonal antibodies (e.g., Cetuximab) or intracellular kinase inhibition using small-molecule TKIs (e.g., Erlotinib, Afatinib).

What is the overarching mechanism of "pathway bypass" shown by tumour cells when EGFR is successfully inhibited?

Tumour cells can reactivate the kinase cascade (specifically ERK) to maintain survival and proliferation, completely in the absence of EGFR activation.