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Terms in this set (26)

-GPCR: A GPCR is a plasma membrane receptor with seven transmembrane helical segments. It is associated with a G-protein that cycles between active, GTP bound form, and inactive or GDP- bound form. There is also an effector enzyme or ion channel that is regulated by the activated G protein.GPCRs are heterotrimeric, meaning they have three different subunits: an alpha subunit, a beta subunit, and a gamma subunit. Two of these subunits — alpha and gamma — are attached to the plasma membrane by lipid anchors

-Three essential components define signal transduction through GPCRs: a plasma membrane receptor with seven transmembrane helical segments, a G protein that cycles between active (GTP-bound) and inactive (GDP-bound) forms, and an effector enzyme (or ion channel) in the plasma membrane that is regulated by the activated G protein. The G protein, stimulated by the activated receptor, exchanges bound GDP for GTP, then dissociates from the occupied receptor and binds to the nearby effector enzyme, altering its activity. The activated enzyme then generates a second messenger that affects downstream targets. A G protein alpha subunit binds either GTP or GDP depending on whether the protein is active (GTP) or inactive (GDP). In the absence of a signal, GDP attaches to the alpha subunit, and the entire G protein-GDP complex binds to a nearby GPCR. This arrangement persists until a signaling molecule joins with the GPCR. At this point, a change in the conformation of the GPCR activates the G protein, and GTP physically replaces the GDP bound to the alpha subunit. As a result, the G protein subunits dissociate into two parts: the GTP-bound alpha subunit and a beta-gamma dimer. Both parts remain anchored to the plasma membrane, but they are no longer bound to the GPCR, so they can now diffuse laterally to interact with other membrane proteins. G proteins remain active as long as their alpha subunits are joined with GTP. However, when this GTP is hydrolyzed back to GDP, the subunits once again assume the form of an inactive heterotrimer, and the entire G protein reassociates with the now-inactive GPCR. In this way, G proteins work like a switch — turned on or off by signal-receptor interactions on the cell's surface.
1. Ligand binds to receptor (example of a ligand could be epinephrine and receptor is adrenergic receptor)
2. G protein is stimulated by activated receptor and exchanged GDP for GTP
3. G protein dissociated from the occupied receptor and binds to nearby effector enzyme
This activated effector enzyme (example could be adenylyl cyclase)
4. The activated enzyme then generates a second messenger that affects downstream targets. (could be cAMP)
6. Downstream targets are activated (example cAMP activated PKA)
7. In the case of PKA (protein kinase A) it phosphorylates other cellular proteins that is the effect of the ligand that bound to original receptor

Details:
-The binding of epinephrine to a site on the receptor deep within the membrane promotes a conformational change in the receptor's intracellular domain that affects its interaction with the second protein in the signal-transduction pathway, a heterotrimeric GTP-binding stimulatory G protein, or Gs, on the cytosolic side of the plasma membrane.
-When the nucleotide-binding sire of Gs (on the alpha subunit) is occupied by GTP, Gs is active and can activate adenylyl cyclase; with GDP bound to the site, Gs is inactive. Binding of epinephrine enables the receptor to catalyze displacement of bound GDP by GTP, converting Gs to its active form. (2). As this occurs, B and gamma subunits of Gs dissociate from the a subunit, and Gsa, with its bound GTP, moves in the plane of the membrane from the receptor to a nearby molecule of adenylyl cyclase (3).
-The Gsa is held to the membrane by a covalently attached palmitoyl group. The association of active Gsa with adenylyl cyclase stimulates the cyclase to catalyze cAMP synthesis (4), raising the cytosolic [cAMP]. This stimulation by Gsa is self-limiting; Gsa is a GTPase that turns itself off by converting its bound GTP to GDP.
--cAMP does not affect phosphorylase b kinase directly. Rather, cAMP dependent protein kinase (kinase A or PKA), which is allosterically activated by cAMP (5), catalyzes the phosphorylation of inactive phosphorylase b kinase to yield the active form. The inactive form of PKA contains 2 catalytic subunits © and 2 regulatory subunits ®. The tetrameric R2C2 complex is catalytically inactive, because an autoinhibitory domain of each R subunit occupies the substrate-binding site of each C subunit. When cAMP binds to two sites on each R subunit, the R subunits undergo a conformational change and the R2C2 complex dissociates to yield two free catalytically active C subunits.
-(6)PKA regulates a number of enzymes. Although the proteins regulated by cAMP-dependent phosphorylation have diverse functions, they share a region of seq similarity around the Ser or Thr residue that undergoes phosphorylation, a seq that marks them for regulation by PKA.
-(7): cyclic AMP, second messenger, is short-lived. It is quickly degraded by cyclic nucleotide phosphodiesterase to 5'-AMP, which is not active as a second messenger.
-Studies have revealed the location of the nucleotide-binding pocket and the interface between Gα and Gβγ subunits. The nucleotide-binding pocket is located between Ras-like domain and α-helical domain of Gα subunit surrounded by four flexible regions (p-loop , switch I, switch II, and switch III).
Ras-like domain hydrolyze GTP and provide binding sites for Gβ subunit. The N-terminus of Gα subunit is reported to be critical for the structure and function of Gα subunit and is myristolylated or palmitoylated suggesting the role of this region in the attachment to the plasma membrane.
In the GTP-bound conformation, the G protein exposes previously buried regions, called switch I and switch II that interact with proteins downstream in the signaling pathway, until the G protein inactivates itself by hydrolyzing its bound GTP to GDP.
The critical determinant of G-protein conformation is the y phosphate of GTP, which interacts with a region called the P-loop (phosphate binding).
In Ras, the y phosphate binding of GTP binds to a Lys residue in the P loop and to two critical residues, Thr35 in switch I and Gly60 in switch II, that hydrogen bond with the oxygens of the y phosphate of GTP. These hydrogen bonds act like pair of springs holding the protein in its active conformation. When GTP is cleaved to GDP and Pi is released, these hydrogen bonds are lost; the protein relaxes into its inactive conformation, burying the sites that interact with other partners in its active state. Ala146 hydrogen bonds to the guanine oxygen, allowing GTP, but not ATP to bind.
-Several signaling proteins contain SH2 domains, all of which bind P-Tyr residues in a protein partner.

-Several signaling proteins contain SH2 domains, all of which bind P-Tyr residues in a protein partner.
One of these target proteins (Fig. 12-6, step 2 ) is insulin receptor substrate-1 (IRS-1). Once phosphorylated on its Tyr residues, IRS-1 becomes the point of nucleation for a complex of proteins (step 3) that carry the message from the insulin receptor to end targets in the cytosol and nucleus, through a long series of intermediate proteins.
First, a P -Tyr residue in IRS-1 is bound by the SH2 domain of the protein Grb2. (SH2 is an abbreviation of Src homology 2 ; the sequences of SH2 domains are similar to a domain in another protein Tyr kinase, Src)
Grb2 also contains a second protein-binding domain, SH3, that binds to regions rich in Pro residues. Grb2 binds to a proline-rich region of Sos, recruiting Sos to the growing receptor complex. When bound to Grb2, Sos catalyzes the replacement of bound GDP by GTP on Ras, one of a family of guanosine nucleotide-binding proteins (G proteins) that mediate a wide variety of signal transductions (Section 12.4).
When GTP is bound, Ras can activate a protein kinase, Raf-1 (step 4 ), the first of three protein kinases—Raf-1, MEK, and ERK—that form a cascade in which each kinase activates the next by phosphorylation (step 5 ). The protein kinase ERK is activated by phosphorylation of both a Thr and a Tyr residue. When activated, it mediates some of the biological effects of insulin by entering the nucleus and phosphorylating proteins such as Elk1, which modulates the transcription of about 100 insulin-regulated genes (step 6 ).
Grb2 is an adapter protein with no intrinsic enzymatic activity. Its function is to bring together two proteins that must interact to enable signal transduction. In addition to its SH2 (P-Tyr binding) domain, Grb2 also contains a second protein-binding domains, SH3, that binds to a proline-rich region of Sos, recruiting Sos to the growing receptor complex. When bound to Grb2, Sos acts as a guanosine nucleotide-exchange factor (GEF), catalyzing the replacement of bound GDP with GTP on Ras, a G protein.
-Cellular responses to dozens of cytokines and growth factors are mediated by the evolutionarily conserved Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway. These responses include proliferation, differentiation, migration, apoptosis, and cell survival, depending on the signal, tissue, and cellular context. JAK/STAT signaling is essential for numerous developmental and homeostatic processes, including hematopoiesis, immune cell development, stem cell maintenance, organismal growth, and mammary gland development Janus kinases (JAKs) were identified through sequence comparisons as a unique class of tyrosine kinases that contain both a catalytic domain and a second kinase-like domain that serves an autoregulatory function. They were functionally linked to STATs and interferon signaling in powerful somatic cell genetic screens. The JAK/STAT cascade is among the simplest of the conserved metazoan signaling pathways. The binding of extracellular ligand leads to pathway activation via changes to the receptors that permit the intracellular JAKs associated with them to phosphorylate one another. Trans-phosphorylated JAKs then phosphorylate downstream substrates, including both the receptor and the STATs. Activated STATs enter the nucleus and bind as dimers or as more complex oligomers to specific enhancer sequences in target genes, thus regulating their transcription

-Binding of erythropoietin (EPO) causes dimerization of the EPO receptor, which allows JAK, a soluble Tyrkinase, to bind to the internal domain of the receptor and phosphorylate it on several Tyr residues. (a) In one signaling pathway, the SH2 domain of the STAT protein STAT5 binds to P-Tyr residues on the receptor, bringing it into proximity with JAK. Following phosphorylation of STAT5 by JAK, two STAT5 molecules dimerize, each binding the other's P-Tyr residue, thus exposing a nuclear localization sequence (NLS) that targets the dimer for transport into the nucleus. In the nucleus, STAT5 turns on the expression of EPO-controlled genes. (b) In a second signaling pathway, following EPO binding and autophosphorylation of JAK, the adaptor protein Grb2 binds P-Tyr in JAK and triggers the MAPK cascade, as in the insulin system
-Many different GPCR receptors that activate different G proteins that can activate other enzymes other than adenylate cyclase. For example, an activated G protein can activate PLC, which is Phospholipase C. PLC can act on phospholipids and hydrolyze them. These are lipids that are anchored to the membrane and contain a phosphate group and sugar group. PLC with cut between the lipids and the phoshphate sugar to produce DAG (diacyl glycerol) and IP3. IP3 (inositol 1,4,5-triphosphate (IP3) is a sugar covered with three phosphate molecules. DAG is glycerol with one empty hydroxyl group and two lipids anchored into the membrane. DAG is able to activate another kinase, PKC. PKC and PLC are both enzymes that add phosphate onto target proteins, but have different targets. IP3 is a second messenger that can cause the release of calcium from intercellular storages from the ER. IP3 binds to a calcium channel on the ER and allows the release of Calcium into the cytoplasm. This causes a higher increase in the Ca2+ concentration in the cytoplasm of the cell all due to the binding a signal molecule to the GPCR. Ca2+ can also stimulate PKC, so PKC is being activated by both DAG and PKC.

-Ca2+ has many important roles as an intracellular messenger. The release of a large amount of free Ca2+ can trigger a fertilized egg to develop, skeletal muscle cells to contract, secretion by secretory cells and interactions with Ca2+ -responsive proteins like calmodulin. To maintain low concentrations of free Ca2+ in the cytosol, cells use membrane pumps like calcium ATPase found in the membranes of sarcoplasmic reticulum of skeletal muscle. These pumps are needed to provide the steep electrochemical gradient that allows Ca2+ to rush into the cytosol when a stimulus signal opens the Ca2+ channels in the membrane. The pumps are also necessary to actively pump the Ca2+ back out of the cytoplasm and return the cell to its pre-signal state
-It is a very important CDK substrate. When DNA damage is detected, the pRb protein participates in a mechanism that arrests cell division in G1. It functions in most cell types to regulate cell division in a response to various stimuli.
The mechanism described below gives the cell time to repair its damaged DNA before it enters into the S phase, which ultimately avoids the potentially disastrous transfer of a defective genome to one of both daughter cells.
-Mechanism:
When pRb is unphosphorylated, it binds to transcription factor E2F. When it is bound to E2F, E2F is unable to promote transcription of a group of genes that are necessary for DNA synthesis. Therefore, the cell cycle cannot proceed from the G1 phase to the S phase. The pRB-E2F blocking mechanism is relieved when pRB is phosphorylated by cyclin E-CDK2, and this phosphorylation happens in response to a signal for cell division to proceed. When the protein kinases ATM and ATR detect DNA damage, they activate p53 to serve as a transcription factor to stimulate the synthesis of p21. p21 inhibits the protein kinase activity of cyclin E-CDK2; therefore, in the presence of p21, pRB remains unphosphorylated and bound to E2F, blocking the activity of this transcription factor, and ultimately arresting the cell cycle in G1.
-This gives the cell time to repair its DNA before entering the S phase, thereby avoiding the potentially disastrous transfer of a defective genome to one or both daughter cells. When the damage is too severe to allow effective repair, this same machinery triggers a process, apoptosis, that leads to the death of the cell, preventing the possible development of a cancer.