mon, april 16th mech of hormone action G-protein cont.

G proteins continued....
back to the diagram from friday....
the receptors that use G proteins as part of the signal transduction mechanism is a serpantine (7 trans membrane domain receptor) a single polypeptide chain that weaves in and out 7 times. each receptor has a particular G protein associated with it. it is called a G protein because it binds GTP or GDP. It is a trimer with alpha, beta, gamma, and bound to GDP and is inactive,once the hormone binds to the extracellular change of the receptor, and that causes a conformation change in the signal transduction domain which triggers a conformational change in the G protein and so GDP dissociates and GTP binds to the alpha subunit splits from the beta and gamma subunits.... now alpha subunit with GTP (active and can regulate other enzyme activity or ion channels open/close in membrane) and the beta/gamma dimer (active too), and also regulate both as well, now. the specific actions of the different active subunits depends upon which G protein you are looking at. the rest of the process is summarized in the handout. (fig) In that case they show what the active alpha subunit can do or beta/gamma subunit can do on a different target protein. the active alpha subunit interacts with some target protein in the plasma membrane, such as an enzyme. the interaction between the active alpha subunit and the target protein either increases or decreases the activity of that target protein. this target protein remains activited or inactivated for as long as the alpha subunit GTP complex is bound to it. the process ends because the alpha subunit has inherent GTPase activity. when the alpha subunit binds to the target protein, it activates the GTPase actity, and it is hydrolyzed into GDP, and eventually recombines with the beta/gamma subunit and reforms the inactive trimer.

this is the general scheme. inthe specificity, there are different alpha, beta, and gamma subunits and there are differet target enzymes. specific serpantine hormone receptors are associated with specific G proteins. these G proteins function as part of the signal transduction pathway in lots of other systems besides just hormone systems. the only ones we are going to talk about are the ones that relate to what we are talking about in this class. there are at least 20 alpha subunits, and we are only interested in 3 of those: alpha f, alpha i, and alpha q. there are not as many different types of beta/gamma dimers. any G protein that contains an f, is GsubF, GsubQ, and GsubI, etc. thyroid stimulating hormone, and TRH both work through this mechanism. The alpha subunit of each G protein determines which signal transduction cascade in the cell is activated or inhibited. Gf and Gi regulate cAMP in the cells, and Gq regulates the phosphoanoxitide pathway.

We will start with Gf and Gi, both of these regulate Gf increases activity of adenylylcyclase activity and Gi decreases adenylylcylase activty. that basic pathway that couples adenylelcyclase to some end product or end event in the cell is summarized in the handout. Adenylylcyclase is a membrane associated enzyme that converts ATP into cAMP. cAMP is a very small molecule, that is considered a second messanger because it can freely difuse inthe cytoplasm of the cell. it is not associated with the membrane necessarily. the role it plays in the cell is that it activates the enzyme protein kinase A. (kinase is an enzyme that attaches phosphates to a protein, such as other enzymes that regulates them) Protein kinase A is a major regulator of other enzymes. those other enzymes regulated then can regulate many other processes in the cell, or a change in the expression of certain genes in the cell, or kinetics of ion opening/closing can change. the last part of the scheme shows that the enzyme phosphodiesterase, then is responsible for breaking down cAMP, and 5prime AMP and then is inactive. the beauty of this scheme is that this allows one hormone molecule to bind with one receptor and activate this pathway and the end result that a billion different reactions of one kind will happen in the cell. 'signal applification'. it is shown on the handout. if you use as an example: epi and it binds to liver cells and triggers breakdown of glycogen into glucose. if one epi binds to its receptor... Gf protein activated.... adenylelcyclase activated... protein kinase A is activated.... number of enzymes activated... and glycogen is broken down into glucose.... 10^8 glucose molecules created from a single epi molecule. in addition to applification this pathway is super rapid. less than a minute to get this going, because all that has to happen is enzymes being phos'd. But if you look at this handout to see that it can also control gene expression, you notice that that times a couple of hours by comparison.

I expect you to know the level of detail that is on the handout.

Gf activate the pathway, and Gi inhibit this pathway. the other pathway that is an issue, is the Gq pathway. if Gq is activated that results in an increase in phospholypase C-beta activity. And the activity what the function of phospholypase C-beta is an enzyme that is associated with the membrane, and within the membrane are phospholipids such as phosphoanostitol,4,5-disphosphate (on the left of handout), the part of this molecule that is slightly more interesting is the anostitol/phosphate group shown in blue and pink. phospholypase C-beta cleaves the phosphoanostitol group away from the rest of the phospholipid, and you are left with is diacylglycerol that is membrane bound (DAG) and inostitol 1,4,5 triphosphate (IP3) which is a mobile molecule in the cytoplasm, like cAMP, so it is considered a second messanger molecule too. DAG and IP3 are both regulatory molecules in the cell. the pathways: either activated either alphaQ subunit or the beta/gamma dimer, activates phospholypase C-beta and phosphoanostitol, 4,5-disphosphate is cleaved to DAG and IP3. IP3 is important because it interacts with the ER which stores calcium. so when it binds to sites on the ER, the calcium channels open and calcium difuses out of the ER into the cytosol. now it can do 2 things: bind to calmodulin which can then regulate other stuff, or calcium and diacylglycerol together convert inactive protein kinase C to active which is like protein kinase A that it regulates in that it regulates the activity of lots of differnt enzymes, and it can regulate gene expression by what it controls. The idea here is that one molecule binds to a recpetor and is bound to a G protein, it is possible for tons of stuff to happen in the cell due to a sincle molecule bound. But it is limited by which enzymes are present. IE: epi binding to a receptor that is linked to Gf in the liver, and the end result is glycogen breakdown to glucose, but if epi binds to pasemaker cells in the heart, and a G protein linked receptor in the heart an end result of destabilization of the heartrate. What effect a hormone has on a cell, depends upon what the signal transduction cascade is linked to. You don't need to know the structures of IP3 or PIP2, but you do need to know all of the pathways as words.

Now where the situation gets confusing, this information is presented so that hormones look like they activate specific things, and not others, but that isnt' true... there are tons of crossovers to other pathways. There are always other minor pathways that are also activated. When there is an increase in the cytosolic calcium concentration that can interact with other signal transduction pathways, so there can be crossover. if you do work in this area it can become a nightmare. so just leave youselves open that there is a lot of complexity.

G-protein linked pathways, or any signal transduction systems... what causes the signaling to end? probably the most obvious thing to happen is tht the hormoenn dissociates from its recptor. when that happens the stimulus is gone. Each hormone will bind to a given receptor only once or twice. a second way a pathway ends is if GTP is hydrolyzed. third is cAMP is hydrolyzed. fourth: calcium is removed from the cytosol. fifth: receptor is phos'd. both of these pathways activated protein kinase A or C. and either of those can turn around and phosphorylate the receptor. which reduces the affinity for the hormone, and reduces affinity for G-protein too. it also can activiate a pathway for endocytosis of the receptor. The most important thing depends upon the target cell. when people talk about receptors being down regulated, is reducing receptors on teh surface in some way.

In a real context TSH works by activating Gs, TRH binds to a receptor that is associated with Gp. The last thing to talk about with respect to hormone mech of action and the hypothylamic /thyroid/pit axis is the mech of action of T3 and T4.

Overview: T3 and T4 have 2 entirely different ways they work on cells. T3 and T4 acting inside a cell as transcriptional activators. Then we will talk about them binding to membrane bound receptors. To talk about their mech of action within the nucleus we have to do an overview of an entire family of hormone receptors. We will talk about the nucleur receptor super-family.

the nucleur rec superfamily. they all work as transcription factors. the hormone binds to a receptor inside the cell and the hormone receptor complex then acts as transcription factor that regulates gene expression. this nucleur super family contains close to 100 members. alot of the AAs seq of them are mRNA and don't know what the ligand is yet. for us whats important that they include receptors for thyroid hormone and for steriod hormones. there are distinct receptors for each steroid class and for thyroid and for vitD3 (also made from chol.). you have a thyroid hormone receptor, estrogen receptors, progestin receptors, glucocorticoid receptors, androgen receptors, and mineralcortacoid receptors. All these different receptors that belong to a super family of receptors because they have a basic similar structure. There is a DNA binding domain, and farther along is a hormone binding domain. the hormone binding domain are relatively specific for the class of hormones. there is an overlap between glucocort. receptors and mineralcort receptors though. the dna binding domain is the area of the molecule that interacts with DNA and so this particular part of the molecule binds to a nucleotide sequence in the dna called the hormone response element. The hormone response element is sometimes called an HRE, and if you want to talk about the specific horomone resposnse element for thyroid is TRE. the actual nucleotide seq to which the different steroids and thyroid hormone bind is very worked out. but you do not need to mem the nucleotide seq at all.

the way these work is that the dna binding domain binds to the hormone response element on the dna and that unit acts in the regulatory region of the gene. when that binding occurs the receptor acts as a transcription factor to regulate transcription of the gene. Now the thyroid method of action is slightly different in the binding details. and thyroids differ as well. See overhead.
General structure for thyroid/steriod hormone receptors... there are different subtypes of thyroid horm receptors and steroid hormone receptors... there are alpha1 THR, beta 1, and beta2 THR, all of these subtypes bind thyroid hormones but some are preferentiall expressed in only certain tissue. beta2 is usually only in the hypthal, and ant.pit. knowing this you can guess that they are the ones thought to mediate the feedback between TRH/TSH/T3/T4. someting in the liver won't be a part of that process.

Another figure from reading nubmer 2 on eres.

thyroid hormone receptors: thyroid hormone stims thermogenin or uncoupling protein in the mitochhondria, which uncouples the proton gradient from ATP. you give more thyroid turns on more thermogenin production (positive feedback). also T3 and T4 inhibit TRH, and TSH, so you add thyroid hormone and it inhibits gene expression, which is harder to visualise the negative feedback. (thermogenin is a protein that functions in the proton chanel in the inner mit. membrane and when it is open it fights the protons that are diffusing down their concentraion gradient and can bypass atpsynthase.)

Lets look at the mechanism that people think thyroid horomones turn on gene expression (see handout, 2 figures) Activating gene expression by thyroid hormones. in the resting state if there is no thyroid hormone present the THR is bound to the TH response element in the DNA, usually as a dimer of 2 THR, or a heterodimer of 1 THR and an RXR receptor (receptor for sys-retinormic acid) these 2 receptors bind the the HResp element and sit there. bound to them are molecules called c0-repressors. they respress transcription. and the corepressors inhibit transcription because they contain dipsome-deacetylase activity. they remove acetyl groups from histones that make up the chromatin. this leads to the hiding/blocking of the transcription start site. if you remember from bio20a: dna doesn't exisst in the nucleus as a naked dna. it is in histones, and is wrapped. the more tight it is the more difficult it is for the transcription factors to find the start sites and to begin. so tighter means less likely a gene will be transcribed. where the thyroid aren't bound to the receptor, the receptor is associated with the corepressors to keep the histones tightly bound around the gene of interest. when T3 is present it binds to the binding site on the thyroid hormone receptor to change the conformation of it and then the corepressor can no longer bind. and so the coactivators are now recruited and are able to bind (they act in the opposite fashion), they contain acetylase activity histones are acetylated and the chromatin opens up so now the transcription machinery can get to the dna and transcription starts. some of the coactivators (nucleur proteins) some of them are histone acetylase enzymes. they add acetyl groups to the histone proteins and the charge on the groups causes the histones to not be so tightly packed and allows easier access for the basal transcription machinery and rna polymerase to come in.

What the thyroid hormone does in this scenario where it increases gene expression is that when it binds to its receptor that causes a conformational change in the receptor, and it releases the corepressor, and leaves, and a coactivator binds instead, acts as a histone acetylase. As soon as you start transcription you can start getting mRHA from this gene.

This is the scheme that people accept as pretty much true. the difficulty comes when you have to use it to explain how T3 and T4 inhibit something. and the option is to turn it on. But in the case of TRH synthesis it is on and we need to turn it off. this is a very active area of research and they don't have it fully figured out. the current model is kind of the opposite of the above... for example, in a neuron producing TRH, where there is not much T3 present, you would have the thyroid hormone recptor diamer bound to DNA, and in that unbound, prerecpetor form, it is able to recruit activators. T3 shows up and binds and the coactivators are replaced with corepressors. the evidence is good that the receptor in both cases there has to be a receptor DNA interaction to get either effect.