Second Messengers

Binding of ligand (L) to G protein-coupled receptor (R) causes association between receptor and heterotrimeric G protein (alpha-beta-gamma subunit). After association, GDP, which is tightly bound to the alpha - subunit in the unstimulated state, dissociates from guanine nucleotide binding site. GTP quickly takes its place.

G-protein coupled receptor signaling (con’t):

Binding of GTP induces a conformational change, causing dissociation of the effector alpha subunit complex and terminates the regular signal. Alpha subunit has GTPase activity, can be turned on by GAP’s, regulatory step where receptor activator can be modulated. If GAP’s are activated and cleaves GTP to GDP and Pi, when this occurs activating mechanism is no longer in effect, and receptor activator is being turned off.

Some receptors can be turned off by GRK (G protein receptor kinase) phosphorlation dependent desensitization (inactivation).

Once GTP cleaved by alpha subunit GTPase activity (maybe enzyme linked) changes from GTP to GDP allows dissociation from effector and re-association with beta-gamma subunit and recycles the protein.

Other mechanisms that are responsible for receptor inactivation, termination mechanisms are very important. 1^st example was GRK (see above) after GRK did its job it can bind beta-arestin allowing receptor to be internalized. Can generate signaling inside cell and then be recycled and reused (dephosporlated in the cell).

There are many families of G protein subunits alpha subunit most studied, some stimulatory, some inhibitory, etc. work by a whole array of mechanisms: cAMP, phospholipids, PIP2, etc.

Activation of alphas lead to increase of cAMP , effector enzyme is adenalin cyclase. cAMP interacts and frees protein kinase A to work in cell and phosporlates (removes block).

Phospholipid Breakdown:

Backbone structure of triglyceride composed of Fatty Acid. Arachidonic acid (important signaling molecule synthesized heats, prostaglandins and many other) in position. Activation of different phospholipase: A - cleaves unsaturated fatty acids in position two releases arachidonic acid. C – cleaves FA in position three linked to cholin, insitol or ethanolamine. Serine release leaves you with diacyleglycerol (DAG) and inositol with phosphate (IP3). This cleavage into DAG and IP3 signaling event to many G protein receptors.

Phospholipase C (PLC) – highly preserved catalytic domain (X and Y), and a varying docking domain that can be activated by different elements, acting on different sites.

Beta-Gamma subunit of protein G can couple with docking unit of PLC this leads to formation of IP3 and DAG. There are several different isoforms of PLC and what it associates with downstream. PLC can also be activated by PTK (phosphotryosine) receptor. PTK receptor auto phospholates itself which then activates PTK and PLC.

These mechanisms work simultaneously making the effect of the ligands more potent. Activation by Ach --- PLC --- IP3 --- Ca++ --- PKC (allows certain kinases to be activated). PKC binds DAG direct activator (some require intracellular Ca++ increase). PKC once activated is another mechanism by which downstream reactions take place (insulin release).

Caveoli – Caveolae are vesicular invaginations of the plasma membrane. The chief structural proteins of caveolae are the caveolins. Caveolins form a scaffold onto which many classes of signaling molecules can assemble to generate preassembled signaling complexes. In addition to concentrating these signal transducers within a distinct region of the plasma membrane, caveolin binding may functionally regulate the activation state of caveolae-associated signaling molecules.

Some hormones use the same mechanism with general intracellular events. Thus it’s a question of specificity which is believed to be achieved by caveoli – one hormone one effect. Caveoli are highly enriched with caveli 1 and 2, it is an area that concentrate hormone action into a location. Receptor can only enter caveoli if ligand has binded to receptor and allow for compartment action they are not random but in substructure that improve downstream specificity. Another mode of action is receptor already in caveloi and can only act once ligand binds.

Variety in structure, regulation and physical distribution allows specificty of overall response: There is a great variety of each molecular component. Each component is represented by several isoforms, Each isoform can be differentially regulated, for example, by phosphorylation or by Ca2+, and be ticketed to a specific place within the cell, presumably because of special signal sequences or domain specificities.

Differential distribution of β-adrenergic receptors: A change in single AA makes a big difference in terms of response, known from studies in lab or human disease (tyrosine not present at specific site) hormone won't exert its effect. Several isoforms to each components and can be regulated allows different down stream targeting.

REMEMBER that we can have simultaneous activation of different mechanisms

Family of hormones that bind membrane and use Kinase / Phoshotase as "second messenger": erythroprotein, EGF, GH, insulin, IGF-I/II, nerve growth factor, oxytocin, prolactin.

LDL, EGF and Insulin (homodimmer) receptors: similar mode of action, receptor has tyrosine kinase activity in cytoplamic portion activated once hormone binds receptor.

Activation of PLC leads to rise in intracellular Ca++ and activation of PKC. Insulin activates several mechanisms for different purposes.

Insulin regulates uptake of glucose into cell by glucose transportase (Glut's). Insulin needs to move glut's to membrane where it can load glucose into the cell. 1^st event depends on tyrosinkinase moiety on intracellular domain once phosphorlation took place it can bind downstream molecules. 1^st is IRS (insulin preceptor substrate), then it's phosphorlated and associated with SH2 which activates PI-3K. Via action of PI-3K we end up with PI3,4,5P this molecule can activate different downstream molecules PKC, PDK (phosphatidle insoitol dependent kinase) allows activation of PKB both required to allow Glut 4 to associate with cell membrane.

IRS are linkers between receptor and signaling best known are IRS1, 2, 3, 4. IRS1 works in muscle, IRS2 works mainly in liver. PI-3K dependent root of activation of PKC and PDK, although other pathways are also activated and are important in activation of Glut4.

Out of PowerPoint presentation:

Two potential insulin receptor–dependent signal transduction pathways. Insulin stimulation results in the activation of a PI 3-kinase–dependent pathway that is necessary but not sufficient to induce GLUT4 translocation. In parallel, the insulin receptor activates an additional pathway leading to Cbl tyrosine phosphorylation through its interaction with the CAP protein. Syn4, syntaxin 4; PI3-K, PI 3-kinase

The phosphorylated insulin receptor binds and phosphorylates IRS proteins and Shc, which bind differentially to various downstream signaling proteins. PI3-kinase is critical for metabolic actions of insulin, such as glucose transport, glycogen synthesis, and protein synthesis, whereas Grb-2/SOS complex, which activates the MAP kinase cascade, is critical in mitogenic response. PI3-kinase probably modulates the mitogenic response as well. Spatial compartmentalization of insulin signal transduction: IRS binds to, and activates the regulatory unit of PI3- kinase and the complex translocates to internal membranes which contain substrate for PI3-kinase and close to GLUT4 which translocates,in response to this process, to the cell membrane

Major signaling pathway of insulin action –

IRS's proteins associated with PI-3K outcome dependents on downstream molecules glucose transport, glycogen synthesis, protein synthesis. Insulin activating the mechanisms of Grb2 and SOS, leading to cell replication (acts as a growth factor).

Insulin Receptor: auto-phosphorlation binds IRS protein and phosphorlate downstream catalytic subunit of PI-3K then the whole complex phosphorylates a specific target in the internal cell structure where Glut 4 resides. Glut 4 is released and traslocated to the cell membrane. Mutation at any point can lead to loss of function.

Hormones with cell membrane receptor for which calcium or/and phosphoinositides serve as second messengers (PLC dependent): α1 adrenergic catecholamines, acetylcholine (muscarinic), angiotensin II, ADH, EGF, GnRH, PDGF (platelet derived growth factor), TRH (tyrotropin releasing hormone --- stimulate TSH).

Regulation of Polypeptide Hormone Synthesis The steroid/retinoic acid/thyroid hormone (nuclear) receptor superfamily example:

Hormone Regulatory Elements (HRE) in the regulatory region of the transcription unit have already been characterized for: glucocorticoid receptor, estrogen receptor, androgen receptor, progesterone receptor, vitamin D receptor, retinoic acid receptor, thyroid hormone receptor (THE NUCLEAR RECEPTOR SUPERFAMILY). Receptor found in cytoplasm once activated it travels to DNA

In each case a DNA element of 8-20 nucleotides confers hormonal regulation to its associated structural region.

Hormones such as steroids and thyroid hormones interact with nuclear receptor proteins which, in turn, interact directly with DNA elements to modulate gene transcription

For example, glucocorticoid ligands bind to inactive glucocorticoid receptor in the cytoplasm which is present in complex with hsp90, hsp70 and other peptides.

Activated receptor- ligand complexes interact as a trans- acting factor to bind to the DNA element GRE.

Cortisol - Steroid Receptor Example: steroid hormone outside cell get through cell membrane (lipophilic) quickly. Cortisol in cytoplasm bind the receptor and causes a conformational change. Receptor is normally in inactive state, bound by hsp70 (heat shock protein) and cannot exert its effect on DNA. Hsp70 is a dimmer linked to receptor preventing downstream action, cortisol induces conformational change, hsp70 dissociates. Complex of receptor and steroid moving freely to nucleus bind hormone response element on DNA (GRE, glucocorticoid response element) induces another conformational change and leads to DNA synthesis.

Phosphorlation must take place for activation

Vitamin D Example: 1alpha,25(OH2)D3 is active form different mode of action.

In inactive form linked as hetrodimer to RXR (retinoic receptor) binding of Vitamin D moves to nucleus still bound VDRE (co-activators) lead to target gene increased transcription rate.

Anatomy of nuclear receptor an d typical gene structure:

LBD – bind steroid (ligand) receptor

DBD – DNA binding domain has zinc fingers and carboxyl terminal with hinge between critical elements at beginning and end AF1 (N-terminus) and AF2 (C-terminus), AF stands for activation functions. Most nuclear receptors superfamily differs in sequence thus binding different activators and co-activators.

Zinc fingers are a part of receptor that is in direct contact with DNA usually has one or two zinc fingers. Zinc fingers are sequence of AA with multiple cystein and histadine this allows for unique folding where Zn binds. There are different arrangements of receptors allowing different association with DNA: monodimmer, homodimmer, RXR heterdimmer. Receptor variations improve specificity, and minimize errors.

Once nuclear receptor bound VDRE (vitamin D response element) with VDR and REX this induces binding of cofactors (activators) in many cases leading to physical folding of area get into direct contact with TATA box. Physical touch of TATA box activating machinery increases transcription rate of RNA polymerase.

In recent years another concept was developed that takes way from the nuclear receptor mode. Particular steroids can also work by interaction with cell membrane directly and generate effects like classical membrane associated receptors. The difference is in the time involved, nuclear activation is a timely process between 30-40min to hours. Cell membrane receptors effects are very rapid, estrogen and testosterone can act within seconds. Therefore there must be another pathway other than the classic nuclear receptor root. For example immediate increase in intracellular Ca++ must be membrane response explains some of the effects. What was initially thought to be two separate modes of action might not be separate but receptors have yet to be identified.

Category: Physiology Notes