Lecture 9: Enzyme Coupled Receptors

Enzyme-coupled rectors and Receptor Tyrosine Kinases

• Enzyme-coupled receptors have a cytosolic domain that has enzyme activity, or directly associates with an enzyme

• Receptor tyrosine kinases (RTKs) is the most common class of enzyme-coupled receptors

• Includes many receptors that control cell growth and behavior

RTKs activate by dimerization/ autophosphorylation

• ligand binding on the extracellular side leads to activation in the following way:

  • unbound RTKs typically exist in an inactive, monomer conformation

  • Ligand binding causes dimerization of the receptors

  • Dimerization brings the two cytoplasmic tyrosine kinase domains into close proximity, so they phosphorylate each other, which leads to further activation

  • Activated domains further phosphorylate themselves, providing docking sites for downstream signaling proteins

  • Unbound RTKs typically exist in an inactive, monomer conformation

  • Ligand binding causes dimerization of the receptors

  • In some cases (EGF receptor), dimerization brings the two kinase domains into an asymmetrical dimer, where one “activator” domain turns on a “receiver” domain, which then phosphorylates both domains

Phosphorylated Tyrosines on RTKs are docking sites for intracellular signaling proteins

• The additional phosphorylated sites on activated RTKs provide high-affinity, specific docking sites for downstream signaling proteins → assembly of an intracellular signaling complex

• Binding alone (conformational change), or subsequent phosphorylation by the RTKs, activates the docked proteins

• Proteins dock to RTKs using SH2 domains or PTB domains

• Some of the docked proteins can be inhibitory, as part of a negative feedback loop

  • addition of ubiquitin to RTKs can cause endocytosis and degradation

Monomeric GTPase Ras mediates most RTK

• Ras is a key oncogene- 30% of tumors have hyperactive mutant forms of Ras

• Ras is a membrane-bound monomeric GTPase

• Ras and Rho family GTPases are activated by Ras/Rho GEFs downstream of RTKs: Ras is activated by Sos. recruited by Grb2 to RTKs

Activated RTKs and Ras are rapidly turned off

• Tyrosine phosphorylations are removed by tyrosine-specific protein phosphatases

• Ras is inactivated by Ras GAP’s

Ras is frequently mutated in human cancers

• Most mutations occur in just 1 of 3 amino acids

• Mutant Ras loses ability to hydrolyze GTP → always in the GTP-bound, ON state

  • Incoming singlas are no longer necessary, Ras signals downstream regardless of growth factor presence

• Results in uncontrolled cell growth

Ras Activates a MAP kinase signaling module

• Mitogen-activated protein kinase module/ pathway (MAP kinase) is a key signaling cascade activated by Ras

• Ras activates MAP kinase kinase kinase

• MAP kinase phosphorylated target proteins important for cell proliferation, including immediate early genes

Scaffold Proteins separate different MAPK modules

• different scaffold proteins are used to assemble different sets of kinases, avoiding unwanted cross-activation

  • humans have 12 MAPKs, 7MAPKKS, and 7 MAPKKKs, that work in at least 5 separate MAPK modules transmitting different signals with different outcomes

Rho GTPases transmit signals to the cytoskeleton

• Rho family GTPases transmit signals from cell-surface receptors to regulate actin and microtubule cytoskeleton function

  • control cell shape, polarity, motility, and adhesion

RTKs also signal through membrane-bound PI(3,4,5)P3

• PIPs, particularly PI(3,4,5)P3, serve as signaling molecules

• Phosphoinositide 3-kinase (PI 3-kinase) is activated by RTKs (and GPCRs) and adds a phosphate at the inositol 3 position

• PI(3,4,5)P3 serves as a docking site for signaling proteins with pleckstrin homology (PH) domains

  • ex. Ser/Thr protein kinase Akt

PI-3-kinase/ Akt is a major survival/ growth pathway

• Pro-survival/ growth signals (ex. insulin like growth factor, IGF) activate RTKs→ PI 3 kinase → PI(3,4,5)p3 → Akt

• Activated Akt phosphorylates many targets at the membrane and in the cytosol, mostly inactivating them

• The end result is pro survival/ gorwth

  • Bcl2 inhibits apoptosis, Bad binds to and inactivates Bcl2

  • Akt phosphorylates and inactivates Bad, turning on Bcl2

PI3k/ Akt activate mTORC1, a key regulator of growth

• mTORC1 (mammalian target of rapamycin) complex controls growth by responding to growth factor signaling (RTKs) and amino acid availability through Rheb and Rag GTPases

• mTORC1 is a protein kinase that phosphorylates many downstream proteins

Some enzyme-coupled receptors act through cytoplasmic tyrosine kinases

• Tyrosine-kinase-associated receptors function similar to RTKs, except the kinase domain is a separate cytoplasmic protein

• REceptors for:

  • antigens

  • interleukins/cytokines

  • integrins

  • some hormones

• Many rely on dimerization for activation

• Some work through the Src family of cytoplasmic tyrosine kinases:

  • contain SH2 and Sh3 domains

  • associated with cytoplasmic side of plasma membrane

Cytokine Receptors activate the JAK-STAT pathway

• Cytokine receptor family binds cytokines (extracellular signaling proteins important primarily in the immune system), some hormones

• Upon ligand binding and dimerization, the associated Janus (JAK) kinases phosphorylate/activate themselves and tyrosines on the receptors

• Signal transducers and activators of transcription (STATs) bind the P-Tyrs via Sh2 domains, are phosphorylated by JAKs, dissociate and form dimers, which serve as transcription activators in the nucleus

TGFbeta proteins activate receptor Ser/Thr kinases

• The transforming growth factor-beta (TGFbeta) superfamily are dimeric signaling proteins that bind and cause multimerization of receptor Ser/Thr kinases

• The receptors phosphorylate themselves and Smad proteins, which trimerize and regulate transcription in the nucleus

• Endocytosis of TGFbeta receptors is important both for further activation (in early endosomes) and for the degradation (through caveolae)