Book Notes: Signaling Part 3

• GPCRs all are from the same family and share characteristics
• sevenpass transmembrane receptors that associate with G proteins
• examples include rhodopsin and olfactory receptors
• signal binds GPCR, GPCR goes through conformational change, activates a bound trimeric G protein, who relays the signal to the rest of the cell
• each GPCR has its own set of G proteins
• the G protein is either normally bound to the receptor or binds after ligand binds to receptor
• G protein has alpha subunit, beta subunit, and gamma subunit (beta and gamma usually go together as the beta-gamma complex)
• in normal state, a is bound to a GDP
• the activated receptor acts like GEF and makes a drop the GDP and get a GTP
• the G protein changes conformation and the subunits can now act on enzymes or ion channels in the plasma membrane to continue the signaling cascade
• when a hydrolyzes the GTP (usually with help from a regulator of g-protein signaling-RGS protein), it becomes inactive
• adenylyl cyclase is one possible target of the G protein
• it makes cAMP from ATP and is membrane-bound
• change of [cAMP] is resisted by cAMP phosphodiesterases (PDEs)
• GPCRs can activate either Gs or Gi to stimulate or inhibit the cyclase
• 2 kinds of toxins target this pathway:
• cholera: bacteria makes enzyme that adds an ADP ribose to the a of a Gs protein, so it can't hydrolyze GTP, so it is continuously on: continuous adenylyl cyclase = continuous high [cAMP] = diarrhea in intestinal cells
• pertussis: bacteria makes enzyme that adds and ADP ribose to the a of a Gi protein, so it can't interact with GPCR to receive a new GTP, so it is continuously off: continuous adenylyl cyclase = continuous high [cAMP] = fluid flooding the lungs
• so with cAMP now present in high concentration, where does the signal go next?
• cAMP activates PKAs (cAMP-dependent protein kinase), a Ser/Thr kinase
• PKA normally exists as 2 regulatory subunits bound to 2 catalytic subunits
• 4 cAMP binding simultaneously to PKA causes the r subunits to release the c subunits, which go to do the phosphorylation
• one of PKA's targets is PDE, which lowers the cAMP, quickly shutting down the signal to a brief local pulse rather than a weak extended signal (like a quick mosquito bite as opposed to the annoying buzz buzz buzzing in your ear)
• A-kinase: another name for the regulatory subunits, help localize PKAs to the right place
• AKAP (A-kinase anchoring protein): an adaptor that tethers the A kinase (and bound c subunits if not activated) to cytoskeleton or organelle membrane or other signaling proteins to make a complex
• so PKA is active, what happens next?
• one option is activation of gene expression
• target gene has a special sequence in the regulatory region of the gene: CRE (cAMP response element)
• the CREB protein recognizes and binds to this sequence
• PKA phosphorylates CREB
• active CREB recruits CBP (CREB binding protein), who stimulates transcription where the CREB is sitting (in that way, the Cre gene is specified by the CREB) 
• G proteins, in addition to adenylyl cyclase, also act on PLC (phospholipase C)
• PLC is activated by the Gq protein, and then cleaves PIP2 into IP3 and DAG
• IP3 is a water-soluble molecule and goes into the cytoplasm
• binds to IP3 receptors at the ER, which are calcium channels
• the channels open and calcium ions flood the cytoplasm
• calcium flood is later reverted by the opening of other calcium channels in the ER and by calcium pumps that expel the ions into the ECM
• DAG is not water-soluble and remains in the membrane, where it is further cleaved into arachidonic acid and later becomes eicosanoids-small lipid signaling molecules (e.g. prostaglandin involved in pain/inflammation)
• DAG also activates PKC: PKC 1st senses the calcium flood and moves to the plasma membrane, where it binds DAG
• triple binding of PKC to calcium, DAG, and phosphatidylserine on the membrane fully activates PKC
• calcium is a universal signal because it CAN cause a sudden flood
• this happens because intracellular [Ca] is kept very very very low, while [Ca] in ER and ECM is very very very high
• as soon as channels open, calcium ions zoom in
• in normal conditions, ATP pumps and antiporters in the plasma membrane and ER membrane expel or suck up calcium out of the cytoplasm
• calcium signals tend to occur in multiple spikes rather than a continuous period of high concentration, because there are so many pumps and other calcium binding proteins
• also, calcium causes its own positive feedback, but too much calcium causes its own negative feedback, producing the oscillation of calcium spikes
• a strong signal produces rapid oscillations, while a weak signal = low frequency of oscillations
• therefore, some calcium sensitive proteins are actually calcium-frequency sensitive: low frequency activates one set of genes, while high frequency activates another
• calmodulin: calcium binding protein that relays the calcium signal
• 2 calcium ions bind calmodulin, causing a conformational change
• the Ca2+/calmodulin complex goes and binds other target molecules, undergoing a second conformational change
• target molecules include the pumps that get rid of calcium
• one important target is CaMk (Ca2+/calmodulin-dependent kinase): CaMk phosphorylates itself and other target molecules
• the autophosphorylation enables CaMk to remain active even when calcium goes away, until phosphatases come by
• CaMk is also the protein that is sensitive to calcium frequency!
• low frequency calcium allows time for the CaMk to lose activity and go back to normal before the next pulse
• high frequency prevents CaMk levels from falling back to zero before the next pulse, so the total CaMk activity goes up, falls a little bit, then goes up even more, falls a little bit, and goes up even even more
• also, CaMk is a multisubunit protein, so a cell can make CaMk's with different subunits to do different things at different frequencies

pg. 903 - 916 (from Chapter 15)