Meeting 19, 20

Hormonal Signaling: control homeostasis in an already developed organism

Developmental Signaling: pattern formation is achieved by inductive signals that change the fate of cells or tissues

signaling center in the most dorsal part of the mesoderm cells in the early frog gastrula; when transplanted in a host embryo, it takes over part of the host embryo and causesthe formation of a nearly conplete new neural & mesodermal axis

-Spemann & Mangold showed that the Dorsal lip functions as an organizer of the embryo, and can even induces the formation of a second embryo when transplanted

• an ion, molecule, or a molecular group that binds to
• another chemical entity to form a larger complex; any molecule, other than an enzyme substrate, that binds tightly and specifically to a macromolecule (usually a protein) forming a macromolecule-ligand complex

*have to bind tightly enough to cause a change but not tightly enough so it never leaves

R + L <---> RL (Receptor + Ligand <--> RL)

K_A = [RL] / [R][L]

K_D = [R][L] / [RL]

[RL]/Rt = 1/(1 + K_D/[L])

-drugs v. ligands: can change ligand….replace it with a drug possibily, and prevent normal binding there; alter funcitons of receptors

• • secretion signaling: produces signals (ex. like those made by dorsal lip) that can travel long distances & act on recipient (ex. pigmented) tissue
• 

vs.

• • direct cell-cell signaling: when tight signaling is needed; molecules move very short range, ex. only talks to neighbor & doesn’t spread
• 

(a) Paracrine: secretory cell releases extracellular signal, which binds to receptor on adjacent target cell, induces change

*Autocrine: target cell is the same as secretory cell; cell secretes extracellular signal, which binds to receptors on ITS p.membrane

(b) Synaptic: nerve cell depolarizes, releases NT into synapse, causes target cell to either depolarize or hyperpolarize, etc. etc.

(c) Endocrine: endocrine gland secretes hormone molecule, which travels via blood stream to distant target cell (binds to rcptr, activates or represses, blah blah blah)

proteins target by signlaing pathways range from:

• (1) metabolic enzymes: alters the target cell's metabolism
• (2) gene regulatory proteins: like a Tx factor; can enter nucleus and change cell's gene expression
• (3) cytoskeletal proteins: can change cell shape or movement

-cell can:

• differentiate

• • divide: signal binds to receptor tells the cell to produce cyclin/CDK to initiate mitosis (cell division)
• * this happens before differentiation; often cells cannot divide once differentiated

• surivive

• • DIE
• - caspase: protease important for apoptosis b/c it degrades protein & DNA

• - Receptor can be:
• • Ion channel-linked (ex. acetylcholine or NMDA rcptr; ligand binding opens channel)
• • Enzyme-linked: triggering a phosphorylation cascade
• • G-protein-linked

• - Activation can involve:
• • Proteolytic processing (ex. Notch: binds to ligand, cuts off the protein inside the cell, protein enters nucleus & activates transcription)
• • Oligomerization (ex. RTKs [receptor tyrosine kinases]; ligand binds to receptor, attracts another ligand, forming a dimer --> causes activation)
• • Conformational change (ex. GPCR)

• example of enzyme-linked receptor: tyrosine kinase
• •adds a phosphate to a tyrosine (*kinases phosphorylate)
• • enzymes here can phosphorylate molecules, ex. serine, tyrosine etc.
• •ligand can bind to 1 receptor, bringing two together cause causing a chain of activation
• • OR binding recruits a protein

• cAMP
• cyclic AMP

• activates protein kinase A (PKA)

• cGMP (cyclic GMP)
• • activates protein kinase G (PKG) & opens cation channels in rod cells

• DAG (diacylglycerol)
• • activates protein kinase C (PKC) by FIRST activating phospholipase C (PLC)
• -PLC is the linker between DAG and PKC

• IP₃ (Inositol-triphosphate)
• • opens Ca²⁺ channels in endoplasmic reticulim

• *relevant here: Ca²⁺ ions also act as 2nd messengers
• 

-often times there is a molecule (or channel) that activates the 2nd messenger, so it can activate it's target molecule

• -cAMP
• • linking enzymes (2)
• (1) adenyl cyclase: makes cAMP from ATP
• (2) phosphodiesterase (enzyme): destroys cAMP

• -cGMP
• • linking enzyme (1)
• (1) guanylyl cyclase: makes cGMP from GTP

• -Ca²⁺
• • linking enzyme (1)
• (1) Ca²⁺ channel

PLC is an enzyme that cleaves phospholipids just before their phosphate group

• one such phospholipids, PIP₂ is cleaved by PLC into DAG & IP₃

• • DAG stays bound to the membrane
• -Ca²⁺ & DAG together activate PKC (protein kinase C)
• -PKC phosphorylates molecules, leading to changes in cell activity

• • IP₃ is released into the cytosol
• - it diffuses through the cytosol & binds to IP₃-receptors, (ex. calcium channels in the endoplasmic reticulum)
• - this causes increase in cytosolic concentration of Ca²⁺, causing changes in cell activity

• • retinoic acid, thyroid hormones and Vitamin D
• are liposoluble steroid hormones that work to maintain homeostasis
• • they're secreted/transported by carriers proteins
• • the (small, hydrophobic) signal enters the cell where it binds to a “nuclear receptor” (ex. RAR)
• • the steroid-receptor complex --> moved into the nucleus, where it activates transcription of genes containing response element (RARE)

• • big rings are hydrophobic & allow transport through membrane
• • all have some charged/hydrophilic activity that allows transport through the membrane (so they don't get stuck there, ex. hydroxyl group, OH-)

one way = GPCR (G-protein coupled receptor); mostly a conformational change in response to binding of a ligand or when light activates a receptor

• • activation of a receptor can just occur by dimerization
• • the binding of 2 ligands to 2 seperate receptors can sometimes cause the complexes to dimerize

• mutation
• • a constitutively active receptor; this can occur when a ligand binding domain is replaced w/ a dimerization binding domain
• -this is the kind of mutation that exists in life & can cause oncogenes

• depending on the membrane potential or ligand channel, K+ channel will open or close

• •this is a 7 g-protein coupled receptor; when ligand binds (red +) changes conformation and causes activation of the G-protein
• -this can be done simply using light

• • all GPCRs have the same orientation in the membrane and contain:
• -7 transmembrane α-helical regions (H1-H7)
• -4 extracellular segments (E1-E4)
• -4 cytosolic segments (C1-C4)
• -C4 (the carboxyl-terminal segment) & the C3 loop (also sometimes the C2 loop) interact w/ G-proteins

• •6-7% of genome is made of GPCR coding genes
• •they're the largest family of cell surface receptors, & largest family of protein in our genome
• -ex. GPCRs code for olfactory genes/olfaction (how you smell)
• •all domains use the same mechanisms to talk to the G-protein
• •they respond to a wide variety of mediators (hormones, NTs, local mediators, epinephrine, serotonin)

• • G-proteins are trimeric, & made of α, β, and γ subunits
• -α & βγ subunits (respectively) are attached to the membrane via covalently bound lipid molecules
• -α subunit: binds & hydrolyzes GTP; codes for GTPase (protein that hydrolyzes, deactivates, GTP)
• -βγ subunit: holds onto the α subunit, brings it to the membrane

• • G-protein is:
• -Inactive when bound to a GDP
• -Activate when bound to GTP
• -in active form (GTP bound to α subunit) --> it to dissociate from βγ subunit

•peak of activation is immediately followed by a drop in activation

• (1) binding of a ligand to a GPCR induces a conformational change in the receptor, activating it
• (2) the activated receptor (+ bound ligand) binds to the G_α subunit
• (3) this causes a conformational change in the G_α subunit, making it release GDP
• (4) now GTP binds to G_α, dissociating it from both the receptor & G_βγ
• (5) the free G_α+GTP now binds to & activates an effector protein (enzyme in the figure)
• -in other news, the ligand bound to the receptor is released when G_α dissociates
• (6) hydrolysis of GTP ends signaling & leads to reassembly of G-protein subunits, returning the system to its resting state
• -binding of another ligand molecule causes the cycle to repeat

G_β is the only subunit of the G-protein NOT attached to membrane via covalent interactions w/ lipid molecules! how cool! remains in place based on association w/ γ subunit

• is a photoreceptor: molecule that can detect light; type of GPCR
• -collects energyof the photon and uses it to change conformation of the receptor
• -its ligand is LIGHT

• 
• • uses ATP to make cAMP; this is done b/c G-protein signaling pathway activates adenylyl cyclase

• • cAMP then activates PKA
• 
• • PKA (protein kinase A) is a serine kinase; cAMP binds to its regulatory subunit & releases active PKA
• • PKA can then go into nucleus & phosphorylate transcription factors (ex: CREB)
• • CREB can only bind to DNA when phosphorylated; activates or represses DNA once bound

• • in this situation, the effector protein is PLC
• • everything in the pathway is the same: ligand binds to receptor, activates G-protein, G_α dissociates, & activates PLC
• (1) once activated, PLC cleaves PIP₂ (phosphatidyl inositol) ester bond, yielding IP₃ and DAG; these 2 molecules induce different pathways
• (2) IP₃ diffuses through cytosol and binds to receptors on ER, opening Ca²⁺ channels
• (3) Ca is released into cytosol
• (4) one of several responses to an increase in cytosolic Ca is recruitment of PKC to the membrane, where it is activated by DAG (DAG activates a kinase)
• (5) the kinase (C) can now alter the activity of numerous enzymes, altering activity in the cell

• • two types of photoreceptors, rods & cones:
• -rods: allow detection of light & are very sensitive
• -cones: detect color; humans have 3 types b/c we are trichromads (red, green, & blue)
• • in the human rod cell, rhodpsin, a light-sensitive GPCR is located in the flattened membrane disks of outer segment
• 
• • opsin has no ligand binding (light, the activator, cannot be 'bound')
• -it's already pre-bound to a 'ligand': 11-cis-retinal
• -when exposed to light, it changes conformation and becomes active or deactivated
• • in the dark: cis isomer; receptor is inactive, ion channels are open
• • in the light: trans isomer; receptor is active, ion channels are closed

• Reviewing the Opsin GPCR when exposed to light (b/c that's when G-protein is activated):
• (1) when opsin is exposed to light, its convered from inactive to active state (all the following is the SAME, binds to inactive GDP bound G-protein, mediates release of GDP & binding of GTP)
• (2) active GTP*G_α binds to PDE, activating it
• -PDE (phosphodiesterase) hydrolyzes cGMP to just GMP
• (3) active PDE turns cGMP to GMP
• (4) the resulting decrease of cytosolic cGMP causes it to dissociate from the NT gated channels in the membrane
• -the channels CLOSE
• (5) membrane becomes 'permanently' hyperpolarized for a lil

- light causes hyperpolarization -

• -activated opsin can be a target for phosphorylation by GRK (G-protein-linked receptor kinase)
• -once tagged with phosphate, this is a signal for arrestin to come bind
• -arrestin binds to opsin & sticks to it, preventing it from subsequently activating a G-protein