Lecture #33: Mechanisms of Hormone Action: Signal Transduction

Read: pg. 672-685

HW: pg. 692 (Study Exercises 5,6) And (Problems 4,5,6,7)

Study Exercises:

5.

1. Adenylate Cyclase (Fig. 21-12; Guided Exploration 19):

A transmembrane protein (receptor) has a high affinity site for ligand (hormone, neurotransmitter or drug) on the outside of the membrane and a G-protein on the inside of the membrane. (The G-protein consists of 3 subunits called stimulatory alpha, gamma and beta and binds GDP)

When a ligand binds to the receptor, it causes the G-protein to bind to the receptor and decreases G-protein affinity for GDP and increases affinity for GTP. Consequently GDP dissociates and GTP associates with the G-protein.

Binding of GTP causes the subunits to dissociate.

The stimulatory alpha subunit diffuses laterally in the membrane until it encounters adenylate cyclase. Binding of the alpha subunit activiates adenylate cyclase which then catalyzes formation of cyclic AMP (cAMP). cAMP then activates cAMP dependent protein kinases or phosphatases... Note that high concentrations of caffeine inhibts phosphodiesterase, the enzyme that converts cAMP back to AMP. Some of the effects of high doses of caffeine are mediated by prolonging the presence of cAMP in the cell.

2. Receptor Tyrosine Kinases (Fig. 21-15; Guided Exploration 20):

When hormones like growth factor bind to their receptors it causes two monomeric receptors (transmembrane proteins) to joint together and form a receptor dimer. When receptors dimerize it activates the tyrosine kinases (located on each of the monomers). The tyrosine kinases then phosphorylate eacho ther (autophosphorylation). The phosphorylated receptor dimer then binds another intracellular protein at a site called the SH2 domain. Once this protein binds to the phosphorylated receptor it leads to a cellular response. For example (see Fig. 21-16) the activated protein (called Grb2) binds Sos which causes Sos to bind and activate a G-protein (called Ras). Activated Ras then binds Raf. Raf is a protein kinase which catalyzes phosphorylation of another protein kinase, phosphorylates another protein kinase etc. (This is called a kinase cascade). Ultimately a protein is phosphorylated that leads to a cellular response. For example, mitogen activating protein kinase moves into the nucleus where it phosphorylates transcription factors that influence gene expression.

3. The phophoinositide pathway (Fig. 21-19)

Phosphatidylinositol-4-5-bisphosphate (PIP2) is a phosphorylated glycerophospholipid that is found in the plasma membrane (inner leaflet).

Ligand binding to a cell-surface receptor activates a G-protein (Gq; consisting of 3 subunits q-alpha, gamma and beta). The q-alpha subunit (in complex with GTP) diffuses latterally in the membrane and activates membrane bound phospholipase C (pg. 224). Phospholipase C (PLC) catalyzes hydrolysis of PIP2 to form diacylglycerol and inositol-1,4,5-triphosphate (IP3) (Fig. 21-18). The charged IP3 molecule is a water-soluble second messenger that diffuses through the cytoplasm to the endoplasmic reticulum. There it opens an IP3 gated Ca2+ transport channel allowing Ca2+ to flow from high concentration (inside the ER) to low concentration (in the cytoplasm). Ca2+ has many effects (eg., it simulates glycolysis and muscle contraction). Ca2+ can also bind to calmodulin which activates calmodulin-dependent proteins.

Meanwhile, the nonpolar diacylglygerol remains embedded in the membrane where it activates protein kinase C to phosphorylate and thereby modulate the activities of several different cellular proteins. (Ca2+ liberated via IP3, facilitates activation of protein kinase C).

6. How do heterotrimeric G-proteins work? (see pg. 675 and Fig. 21-12)

Heterotrimeric G-proteins bind GTP and GDP and consist of 3 subunits (alpha, beta and gamma). When GDP is bound to one of the subunits (G-alpha), the G-protein is turned off (in its inactive form). When a ligand (hormone, neurotransmitter or drug) binds to its receptor, the G-protein associates with the intracellular portion of the receptor. (These receptors are integral transmembrane proteins; Fig. 21-10. The part of the receptor located outside the cell interacts with the ligand; the part of the receptor inside the cell interacts with the G-protein). When the G-protein associates with the receptor GTP replaces GDP. Binding of GTP to the G-protein turns it on. Actually the subunits of the G-protein dissociate and float around in the membrane until they contact someother molecule and turn it on. For example, one of the subunits (stimulatory alpha subunit) activates adenylate cyclase. Adenylate cyclase then catalyzes formation of cAMP which can activate or inactivate protein kinases and phasphatases. The G-alpha subunit then catalyzes the hydrolysis of GTP to form GDP. This turns the G-protein off by causing the G-protein subunits to reassemble. If you add a GTP analog (like GTP-gamma-S) that can not be hydrolyzed it will prolong the activity of the stimulatory G-alpha subunit.