Question

1. Steroid medications can usually be applied to the skin, whereas most other medications must be swallowed or injected. Using what you know about hydrophilic and hydrophobic ligands, explain why there is this difference. In your answer, make sure you describe in detail the pathway that a steroid and non-steroid medication would take to elicit their response.

2. You are interested in studying a new membrane receptor that you have identified, and think that it might be a receptor tyrosine kinase or G-protein coupled receptor. Describe the different transduction pathways downstream of these receptors, and identify three transduction components that could be used to distinguish between the two pathways.

Answer 1

1. Steroids are hydrophobic ligands. Hence, they will easily pass through the hydrophobic barrier of the skin. Steroids, that are cyclic structures, bind to intracellular receptors that are present in the nucleus or cytoplasm. Steroids have to be therefore internalized through the plasma membrane. They travel through skin also bound to plasma proteins as they are insoluble in blood. Thus, steroids can easily pass through the cell membrane, bind to intracellular receptors in cytoplasm, cause their dimerization and transport across the nuclear membrane. The steroid-steroid receptor complex then binds to response elements on DNA and induces transcription of target genes. Skin can also metabolize tropical corticosteroids thereby causing increased absorption of the drugs.

On the other hand, water soluble non-steroidal drugs are repelled by the hydrophobic transmembrane region of the cell membrane of the skin. They are water soluble and hence can be easily transported via blood and extracellular fluid. These hydrophilic water soluble compounds bind to cell surface receptors as they cannot pass through cell membrane. This binding induces activation of secondary messenger which then activate signaling events in the cytoplasm and nucleus. Hence these drugs must be injected while steroidal drugs can be applied to skin.

2. Receptor tyrosine kinases bind to ligands via their extracellular ligand binding domain. This binding causes receptor dimerization via transmembrane region. As a result, there is auto-phosphorylation of tyrosine residues due to tyrosine kinase intracellular domain. The phospho-tyrosine residues act as docking sites for adapter protein that have SH2 or PHB domain to cell membrane. The adapter proteins bind to guanosine exchange factors such as Sos, which then replace GDP on Ras with GTP. Active Ras-GTP then activates Raf by phosphorylation. Phospho-Raf will activate MEK1/2 which then phosphorylates ERK1/2. ERK1/2 translocates to nucleus and binds promoters of target genes such as c-myc, c-fos and increases transcription to induce cell proliferation. Ras-GTP can also activate PI3K which breaks PIP2 in cell membrane to PIP3. PIP3 phosphorylates AKT. Phospho-AKT activates mTOR or cyclin D. The mTOR can then activate transcription and translation of target genes, resulting in increased cell division. Apart from these signaling pathways, there are other signaling pathways involved.

G protein coupled receptors bind to ligands such as adrenaline, noradrenaline and activate the G-protein bound to GPCR. The G protein can be stimulatory or inhibitory. The Alpha subunit of G protein dissociates from Gabg trimer and binds GTP. The Ga-GTP activates adenylyl cyclase to form cAMP. Protein kinase A is activated by cAMP, which then phosphorylates proteins such as CREB. CREB is phosphorylated and translocates to nucleus where it binds to promoter of target genes and activates transcription. In the inhibitory pathway, the Gia can inactivate adenylyl cyclase to inhibit signaling The Gq subunit will activate phospholipase C which breaks down PIP2 to IP3 and DAG. This leads to increase release of calcium from endoplasmic reticulum and downstream calcium signaling events.

Difference between GPCR and RTK:

1. GPCR has 7 transmembrane domains while RTK have one transmembrane domain. RTKs can stimulate 10 or more signaling pathways. GPCR signaling involved lesser signaling events.

2. GPCR undergoes dimerization upon ligand binding. RTK does not show receptor dimerization.

3. GPCR cause hydrolysis of the G alpha subunit from the Gabg trimer (G protein) attached to the receptor. RTKs induce auto-phosphorylation of tyrosine residues on the receptor.

4. GPCR activate adenylyl cyclase and phospholipase C beta. RTKs activate Ras, which Activates PI3K or MAPK pathway. It also activates JAK-STAT, phospholipase C signaling as well.

5. GPCR indirectly activates signaling via the G protein. In RTK, there is direct activation of the pathway on the secondary messenger itself.