Endocrinology/Objectives/Lecture 14

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Contents 1 Hormone-induced second messengers 1.1 Discuss the role of cAMP-dependent protein kinase in glycogen metabolism. 1.2 Explain the concept of amplification of hormone signal. 1.3 Describe the mechanisms of desensitization of β-adrenergic receptor. 1.4 Describe the phosphoinositide signaling cascade. 1.5 Understand the role of PI-3 kinase in the generation of DAG and IP3. 1.6 Describe the relationship between DAG and protein kinase C. 1.7 Recognize the relationship between Gq protein, PLC, DAG, IP3, Ca2+, and PKC. 1.8 Explain the use of phorbol esters. 1.9 Describe the mechanism of action of calcium as a second messenger. 1.10 Describe the synthesis of cGMP. 1.11 Know the ligands and structures of guanylyl cyclase receptors. 1.12 Know the synthesis and mechanism of action of nitric oxide. 1.13 Explain the G protein-coupled receptor-mediated activation of CREB. 1.14 Name hypothalamic releasing hormones and anterior pituitary hormones. 1.15 Understand the roles of G protein-coupled receptors and second messengers in signal transduction mediated by hypothalamic releasing hormones. 1.15.1 CRH 1.15.2 TRH 1.15.3 GHRH

Hormone-induced second messengers

Discuss the role of cAMP-dependent protein kinase in glycogen metabolism.

In general, enzymes required for glycogen phosphorylation (breakdown) are activated by phosphorylation. Those required for glycogen synthesis are activated by dephosphorylation. Thus, protein kinase A stimulates glycogen breakdown and inhibits glycogen synthesis.

Stimulation of glycogen phosphorylation

PKA → ↑GPK → ↑GP → ↑glycogen phosphorylation

Inhibition of glycogen synthesis

PKA → ↓PP, ↓GS → ↓glycogen synthesis

Explain the concept of amplification of hormone signal.

Binding of a single hormone molecule to its receptor can activate multiple downstream messengers. For example, a G protein-coupled receptor may activate up to 100 G_s proteins, which in turn activates a single molecule of adenylyl cyclase (AC). Each AC may produce up to 40 molecules of cAMP, which go on to activate PKA, etc. At each stage of the signaling pathway, the signal may be amplified tenfold. Ultimately, very low levels of hormones can cause dramatic changes in concentrations of second messengers.

Describe the mechanisms of desensitization of β-adrenergic receptor.

β-Adrenergic receptor desensitization may be mediated by the withdrawal of receptors from the cell membrane or by reducing their ability to participate in signaling cascades.

Removal of β receptors is regulated by β-adrenergic receptor kinase (BARK). When ligand is bound to the β-AR, BARK associates with membrane-bound G_βγ subunits. BARK phosphorylates certain serine residues on the cytosolic face of β-AR, which may now bind β-arrestin. β-Arrestin prevents β-AR from interacting with G proteins. In addition, the β-arrestin:β-AR complex is removed from the membrane by clathrin. Clathrin polymerization leads to the formation of coated endocytic pits and vesicles. Inside the vesicles, β-adrenergic receptors are dephosphorylated and eventually reinserted into the membrane (resensitization).

Alternatively, β receptors may be desensitized by PKA. Prolonged activation of PKA leads to phosphorylation of serine/threonine residues on the receptor's cytosolic domain (Presumably these residues are distinct from the residues phosphorylated by BARK.) Phosphorylation produces a receptor that may still bind epinephrine but can no longer lead to the activation of adenylyl cyclase.

Describe the phosphoinositide signaling cascade.

• Hormone binding → G_q protein activation
• ↑G_q → ↑PLC → ↑DAG, ↑IP₃
  • ↑IP₃ → ↑[Ca²⁺]
  • ↑DAG, ↑[Ca²⁺] → ↑PKC

Understand the role of PI-3 kinase in the generation of DAG and IP₃.

PI-3 kinase is both a serine/threonine kinase and an phosphoinositide kinase. Like PLC, it is regulated by both G protein-coupled receptors and by receptor-associated tyrosine kinases (RTKs). PI-3 kinase participates negatively in the generation of DAG and IP₃ second messengers by withdrawing intermediates necessary for their synthesis. For example, the conversion from PI to PIP to PIP₂ is required for the generation of DAG and IP₃. PI-3 kinase phosphorylates PI, PIP, and PIP₂, making them unavailable for DAG and IP₃ synthesis.

Describe the relationship between DAG and protein kinase C.

Membrane-bound DAG is required for the activation of PKC. PKC also requires Ca²⁺ for activation.

Recognize the relationship between G_q protein, PLC, DAG, IP₃, Ca²⁺, and PKC.

G_qα activation activates PLC, which cleaves PIP₂ into DAG (which stays in the membrane) and IP₃ (a freely diffusable second messenger). IP₃ binds and activates calcium channels that release Ca²⁺ from intracellular stores such as endoplasmic reticulum. DAG and Ca²⁺ are coactivators of PKC, which is a protein kinase capable of mediating further downstream events (e.g. growth and proliferation).

Explain the use of phorbol esters.

Phorbol esters are synthetic compounds that mimic DAG in their ability to activate PKC. Since PKC is involved in growth and proliferation, phorbol esters such as myristoyl phorbol acetate are capable of producing tumors.

Describe the mechanism of action of calcium as a second messenger.

Calcium's effects (most, but probably not all) are mediated through the small protein calmodulin. Each molecule of calmodulin binds four Ca²⁺ ions (with aspartate, glutamate, and asparagine residues), producing a CaM complex with an altered conformation capable of driving downstream events. Examples include the activation of cAMP phosphodiesterase, calcium/calmodulin-dependent protein kinase (CaM kinase), and glycogen phosphorylase kinase (GPK).

Describe the synthesis of cGMP.

cGMP is synthesized from guanylyl cyclase (GC) which uses GTP as its substrate.

Know the ligands and structures of guanylyl cyclase receptors.

Two isoforms of guanylyl cyclase

Receptor type	Ligand	Structure	Action
Membrane-bound	Atrial natriuretic factor Guanylin Endotoxin	Membrane-bound Cytoplasmic C-terminal	Atrial natriuretic factor Renal collecting ducts (↑Na+ excretion) Vascular smooth muscle (vasodilation) Guanylin and bacterial endotoxins Intestinal epithelium (regulate Cl- secretion)
Cytosolic	Nitric oxide	Heterodimer	Smooth muscle cells of heart and vasculature

Know the synthesis and mechanism of action of nitric oxide.

Nitric oxide is synthesized from arginine and oxygen by calcium-dependent nitric oxide synthase (NOS). NO is readily diffusable and acts in a paracrine manner. Its half-life is 2-30 minutes because it is oxidized very rapidly to nitrite or nitrate.

In endothelial cells, acetylcholine promotes calcium influx. Calcium binds calmodulin and the complex activates NO synthase. NO diffuses to adjacent vascular smooth muscle cells, where it activates guanylyl cyclase, causing cGMP levels to rise. cGMP results in muscle relaxation and vasodilation. These effects can be mimicked by nitroglycerin tables (used for treating angina).

NO also facilitates synaptic communication in the CNS.

Explain the G protein-coupled receptor-mediated activation of CREB.

A hormone (e.g. epinephrine) binds its receptor (e.g. β-AR), which is coupled to a G_s protein. Activation of G_s activates adenylyl cyclase, which in turn causes a rise in intracellular cAMP. cAMP activates protein kinase A, which can translocate to the nucleus and phosphorylate and activate CREB.

CREB may also be phosphorylated by CaM kinase and PKC through the PLC/DAG/IP₃/Ca²⁺ signaling cascade.

Name hypothalamic releasing hormones and anterior pituitary hormones.

Hypothalamic releasing/inhibiting hormone	Anterior pituitary hormone
GHRH	GH
Somatostatin	↓GH
TRH	TR
CRH	ACTH
DA	↓PRL
GnRH	LH, FSH

Also see objectives from lectures 1 and 2.

Understand the roles of G protein-coupled receptors and second messengers in signal transduction mediated by hypothalamic releasing hormones.

CRH

1. CRH binds receptor
2. Activate G_s protein
3. Activate adenylyl cyclase
4. Elevate cAMP
5. Activate PKA
6. Phosphorylate proteins
   • Release secretory granules containing ACTH, β-LPH, et al
   • Enhance POMC synthesis

TRH

1. TRH binds receptor
2. Activate G_q protein
3. Activate PLC
4. Cleave PIP₂ into DAG and IP₃
5. Release intracellular Ca²⁺
6. Activate PKC
7. Phosphorylate proteins
   • Release TSH secretory granules
   • Enhance expression of TSH

GHRH

• Same G_s protein-coupled pathway as CRH, leading to the release of GH
• Somatostatin (SS) inhibits GH release via G_i protein