Lecture
#4: Hormone Action: Signal Transduction,
Part I
I. G-PROTEINS (guanine triphosphate binding proteins) (Fig. 4A) (Fig.3.7)
1. G-proteins are highly homologous heterotrimeric proteins.that associate with GPCRs.
2. GPCRs make up a superfamily of more than 1,000 molecularly distinct receptors that utilize G-protein-mediated signal transduction to transmit signals across the plasma membranes of various cells.
(a) GPCR ‘ligands’ can be hormones, neurotransmitters, opsins. odors, or inflammatory agents.
(b) about one-half of all modern medicinal drugs target GPCRs.
3. The rule is that ligand/receptor coupling activates GTP-binding proteins (i.e., G-proteins).
4. G-proteins function, ultimately, to transfer the ligand signal to kinases and/or to ion channels.
5. The trimeric G-proteins are composed of a-, b-, and g-subunits.
6. Activated (oftentimes, ‘phosphorylated’) receptors enzymatically activate G-proteins.
7. G-protein activation involves: (Fig. 4A)
(a) replacement of GDP on the a-subunit with GTP (this is not exactly phosphorylation)
(b) dissociation of the a-subunit from the bg-dimer.
8. Usually the a-subunit (20+ distinct varieties now found) determines the kind of cytosolic reaction, but bg-dimers (4+ varieties) can also have functions, such as PLC activation (and, they act with Mg++).
9. Activation of a-subunits cause one or more of the following events:
a. activation of adenylate cyclase ® cAMP formation ® protein kinase A (PKA).
b. activation of phospholipase C (PLC) and phospholipase A2 (PLA2).
c. indirect activation of ion channels in the plasma membrane.
10. There are a number of different categories of G-proteins, including:
a. Gs-proteins, which activate adenylate cyclase and generate cyclic AMP:
(i) cAMP generates protein kinase A (PKA) and activates Ca++ channels/
(ii) PKA phosphorylates proteins such as cAMP-responsive element-binding protein (CREB)
(iii) CREB is a transcription factor that interacts with CRE (a DNA) to promote transcription.
b. Gi-proteins, which inhibit adenylate cyclase, activate K+ channels and inactivate PKA. (Fig.3.7)
c. Gq-proteins, which activate PKC, PLC and PLA2.
d. Gao-proteins activates a specific PLC to release phosphoinositide.
e. Go-proteins, which activate both Ca++ and K+ channels, especially in neurons.
f. Gt-protein is the retinal G protein that activates guanosine 3’,5’-monophosphate-specific phosphodiesterase.
11. Keep in mind that a single receptor can activate several G-protein molecules.
12. G-protein activation is a cyclic phenomenon that depends on ligand-driven receptor activity.
II. ACTIVATION OF ADENYLATE CYCLASES AND CYCLIC AMP
1. There are a number of different adenylate cyclases (ACs)
2. Adenylate cyclases catalyzes ATP to cAMP (Fig. 3.6).
3. cAMP is rapidly metabolized to AMP by phosphodiesterases (Fig. 3.6).
4. Phosphodiesterases are inhibited by caffeine, theophylline, and theobromine (Fig. 3.9).
5. cAMP releases regulatory subunits to activate protein kinase A. (PKA subunits) (Fig. 3.7.1).
6. (NOTE: Phosphorylation can sometimes inactivate enzymes.)
7. cAMP can stimulate certain steroid synthesis, such as progesterone synthesis.
8. cAMP also is associated with the gating of ion channels (as discussed later).
9. Remember that cAMP is inactivated by ligands that couple with Gi-proteins.
III. MEMBRANE PHOSPHOLIPIDS AND THE PHOSPHOLIPASES (Fig. 4.B.A.1)
A. PHOSPHOLIPASE-C
AND HYDROLYSIS OF PHOSPHOINOSITIDES
1. Some receptor-associated G-proteins can activate phospholipase C.
2. Specifically, PLC hydrolyzes membrane phosphatidylinositol 4,5-biphosphate (PIP2) to form two bioactive products, namely diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3) (Fig. 4.B.A.1).
a. DAG is a membrane bound diglyceride that activates PKC. (Fig. 3.12) (Fig. 4.C)
b. IP3 is a cytosol soluble sugar that releases Ca++ from the ER to activate PKC. (Fig. 3.12)
3. PKC is thought to phosphorylate many different cytoplasmic proteins.
(NOTE: At this point, might try a review of this signal transduction process.)
B. CALCIUM IONS AND THE
FORMATION OF A PROTEIN KINASE
1. Ca++ serves as a special "second messenger" to activate another protein kinase, Kcam
2. Cytosolic Ca++ increases not only by the action of IP3 on the ER, but also by ion gating at the plasma membrane and influx from the extracellular fluids. (Fig. 4.D)
3. The action of cytosolic Ca++ is mediated by a Ca++-binding protein calmodulin (Fig. 4.C).
4. Calmodulin undergoes a conformational change in the presence of Ca++.
5. This leads to activation of a calmodulin-dependent protein kinase (Kcam). (Fig. 4.C)
6. Like PKC, Kcam is thought to regulate many different cytoplasmic proteins.
(NOTE: At this point, you might find a
review of second messengers to be helpful.)
C. SUMMARY OF PKA, PKC, AND Kcam FORMATION AND ACTION (Fig. 4.C)
1. Kinases ( along with phosphatases) regulate phosphorylation (maybe thousands of kinases).
2. Phosphorylation processes involve the flow of energy via phosphate bonds in an organized pattern through a number of effector enzymes and regulatory proteins that are sequentially energized to undergo conformational changes that cause characteristic cell responses.
3. Phosphorylation is a reversible covalent reaction (Fig. 3.17).
4. Kinases primarily phosphorylate serine and threonine, along with tyrosine. (Fig. 4.C)
5. Kinases usually span the membrane, or are on its inner surface.
6. Kinases are sometimes referred to as "third messengers" in signal transduction processes.
D. PHOSPHOLIPASE-A2 AND THE FORMATION OF EICOSANOIDS
AND PAF
1. Phospholipase A2 (PLA2) acts mainly on the sn-2 carbon of phosphatidyl compounds to release arachidonic acid and the formation of a number of different bioactive eicosanoids. (Fig. 4.B.A.1)
2. Arachidonic acid is metabolized into three major groups of bioactive compounds: (Fig. F)
a. Cyclooxygenase pathway leads to the formation of the prostaglandins (PGs). (Fig. 3.13)
(i) PGE2 is a vasodilator (causing local hyperemia), and it stimulates adenylate cyclase.
(ii) PGI2 (prostacyclin) is also a vasodilator, and a potent inhibitor of platelet aggregation.
(iii) PGF2a has unclear functions, but it might activate PKC.
(iv) TXA2 (thromboxane), from platelets, increases clotting, and causes vascular constriction..
b. Lipoxygenase pathway leads to leukotrienes and lipoxins.
(i) leukotriene B4 is leukotactic.
(ii) while leukotrienes C4, D4, & E4 are smooth muscle stimulants.
(iii) lipoxin A4 is a potent inducer of angiogenesis.
c. Cytochrome P450-dependent monooxygenase pathway gives rise to bioactive epoxides that can (along with the other products of arachidonic acid) regulate ion gating.
3. These products of arachidonic acid are sometimes referred to as "fourth messengers".
(NOTE: However, your book refers to them as “second messengers.”)
4. Platelet-activating factor (PAF) is another derivative of the action of PLA2 on membrane phospholipids. After removal of arachidonic acid from the sn-2 position, the second step involves the addition of an acetyl group (by acetyl transferase) to a glycerophosphocholine compound. (Fig. 4.B.B.1).
5. PAF promotes inflammation and stimulates more IP3 and DAG.