Endocrinology/Objectives/Lecture 15

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Contents 1 Receptor tyrosine kinases 1.1 Discuss the events that occur after binding of a ligand to a receptor tyrosine kinase. 1.2 Compare and contrast the G proteins and Ras. 1.3 Discuss the role of adaptor proteins in receptor tyrosine kinase-initiated signaling. 1.4 Describe the relationship between Ras, Raf, and MAP kinase. 1.5 Describe the RTK-initiated PI3-kinase pathway. 1.6 Know the importance of RET receptor tyrosine kinase in endocrine neoplasms. 1.7 Compare and contrast the insulin receptor-activated MAPK and PI3-kinase signaling pathways. 1.8 Know the correlation between the metabolic effects of insulin and the insulin-initiated signal transduction pathway. 1.9 Know the correlation between the metabolic effects of glucagon and the glucagon-initiated signal transduction pathway.

Receptor tyrosine kinases

Discuss the events that occur after binding of a ligand to a receptor tyrosine kinase.

1. Hormone binds receptor
2. Receptor dimerizes
   • Receptor autophosphorylates
   • Autophosphorylation produces SH2 domain-binding sites (phosphorylated tyrosine residues)
3. Grb2 adaptor protein binds receptor via its SH2 domain
4. Grb2 attracts Sos (a GEF, guanosine exchange factor) via Grb2's two SH3 domains
5. Sos activates Ras by facilitating the conversion of Ras.GDP (inactive) to Ras.GTP (active)
6. Ras activates downstream Raf
   • Ras' intrinsic GTPase hydrolyzes GTP to GDP after a short while
7. Raf phosphorylates and activates MEK
8. MEK phosphorylates and activates MAPK
9. MAPK phosphorylates cytosolic and nuclear proteins, some of which are transcription factors that alter gene expression

Compare and contrast the G proteins and Ras.

Common features

• Both have intrinsic GTPase activity
• Both are active in the GTP-bound form and inactive in the GDP-bound form

Differences

• Ras is not directly linked to its receptor
• Dissociation of GDP from Ras is very slow; its activation requires a guanosine exchange factor (GEF), e.g. Sos
• Ras' intrinsic GTPase is very slow; Ras' inactivation requires a GTPase activating protein (GAP)

Discuss the role of adaptor proteins in receptor tyrosine kinase-initiated signaling.

Adaptor proteins provide a link between RTKs and Ras. In particular, autophosphorylation of the RTK produces SH2 domain-binding sites (phosphotyrosines) to which the adaptor protein Grb2 can bind. In addition to having an SH2 domain to bind receptor, Grb2 also has two SH3 domains, with which it attracts Sos.

Describe the relationship between Ras, Raf, and MAP kinase.

Ras is effectively a monomeric G protein that can activate Raf. Raf is a serine/threonine kinase that phosphorylates and activates MEK. MEK is a dual serine/threonine and tyrosine kinase capable of phosphorylating and activating MAPK. Because of the relationship between these kinases, Raf is considered a MAP kinase kinase kinase (MAPKKK) and MEK, a MAP kinase kinase (MAPKK).

Activated Raf is membrane-bound due to its association with Ras. Similarly, MEK is membrane-bound due to its association with Raf. MAPK, while it is activated by membrane-bound enzymes (e.g. MEK), can freely diffuse around the cytosol and may even enter the nucleus to regulate gene expression through transcription factor phosphorylation.

Describe the RTK-initiated PI3-kinase pathway.

1. Hormone binds RTK
2. RTK activates by dimerization and autophosphorylation
3. RTK phosphorylates insulin receptor substrate-1 (IRS-1)
4. IRS-1 contains SH2-binding domains which recruit the regulatory p85 domain of PI3-kinase to the membrane
5. PI3-kinase phosphorylates PIP₂, converting it to PIP₃
   • PTEN (a 3-phosphatase tumor suppressor) antagonizes the generation of PIP₃
6. PIP₃ recruits AKT (a serine/threonine kinase, also called PKB), PDK1, and PDK2 to the membrane
7. PDK1 and PDK2 phosphorylate and activate AKT
8. AKT phosphorylates many downstream effectors involved in growth, proliferation, and cytoskeletal rearrangement (e.g. NF-κB)

Know the importance of RET receptor tyrosine kinase in endocrine neoplasms.

RET receptor tyrosine kinase has a single transmembrane domain, a cysteine-rich extracellular domain, and an intracellular kinase domain. Its activation requires ligand binding (by any of GDNF, NTN, ART, and PSP) and coactivation (by any of GFRα1–4). The sequence of activation includes ligand binding to a coactivator, followed by binding of the ligand:coactivator complex to the RET RTK, causing its dimerization and autophosphorylation.

Activated RET RTK directly activates PLCγ and indirectly activates both PI3-kinase (via a Shc-Grb2-Gab1 signal) and MAPK (via the Grb2-Sos-Ras-Raf-MEK cascade). PLCγ activation results in calcium release, MAPK activation results in cell growth and differentiation, and PI3-kinase enhances cell motility and supports cell survival.

Gain-of-function mutations in the RET RTK gene are associated with certain cancers, specifically multiple endocrine neoplasia type 2 (MEN2 A/B) and familial medullary thyroid carcinoma (FMTC). MEN2 is autosomal-dominant and is associated with medullary thyroid cardinoma, pheochromocytoma, and hyperparathyroidism. Loss-of-function mutations in RET RTK are associated with Hirschsprung disease, which is characterized by aganglionosis of the gut and absence of hindgut innervation.

Compare and contrast the insulin receptor-activated MAPK and PI3-kinase signaling pathways.

Common features

• Both may be initiated by insulin binding to its receptor
• Both pathways proceed via the phosphorylation of IRS-1
• Both pathways interact with a number of downstream effectors to alter cell growth and promote cell survival

Differences

• PI3-kinase is activated in the IRS-PI3K-PI3-PDK1-AKT pathway
• MAPK is activated by the IRS-Grb2-Sos-Ras-Raf-MEK-MAPK cascade
• The MAPK cascade ultimately alters gene expression
• The PI3-kinsae cascade ultimately alters glucose metabolism (e.g. regulating glycogen synthesis)

Know the correlation between the metabolic effects of insulin and the insulin-initiated signal transduction pathway.

In responsive tissues, the binding of insulin to its receptor activates AKT via the PI3-kinase cascade described previously. (Ligand binding initiates autophosphorylation and activation of the insulin receptor, which phosphorylates IRS-1, producing SH2 domain-binding sites. IRS-1 recruits PI3-kinase through PI3K's SH2 domain, and PI3K converts membrane-bound PIP₂ to PIP₃ by phosphorylation at the 3-position. PIP₃ recruits AKT [PKB] in addition to its activators, PDK1 and PDK2, to the membrane.) Activated AKT phosphorylates and inactivates glycogen synthase kinase-3 (GSK3), stabilizing the unphosphorylated, active form of glycogen synthase. This accelerates the synthesis of glycogen from glucose.

AKT also promotes the insertion of Glut4 channels into the basolateral membrane to facilitate the entry of glucose into responsive cells.

Know the correlation between the metabolic effects of glucagon and the glucagon-initiated signal transduction pathway.

Binding of glucagon to its receptor results in activation of a G_s protein, which activates adenylyl cyclase and results in a rise in intracellular cAMP. cAMP activates PKA, which phosphorylates and inactivates protein phosphatase and glycogen synthase, inhibiting glycogen synthesis. In addition, PKA also phosphorylates and activates glycogen phosphorylase kinase, which phosphorylates and activates glycogen phosphorylase. Glycogen phosphorylase frees glucose from glycogen stores.