1. a) G protein-coupled receptors (GPCRs), contain transmembrane helices, and their extracellular pats can be glycosylated. GPCRs are divided into two groups that are made up of alpha, beta and gamma subunits. They contain two highly conserved cysteine residues that can form disulfide bonds, to stabilize the receptor structure. They also include seven transmembrane helix proteins (channelrhodopsin) that may contain ion channels within their protein. Its atomic coordinates usually define the protein part of the GPCPs.
2. b) False. Two main factors determine the sensitivity of a cell towards an agonist peptide, the first being the binding affinity between the peptide agonist and its specific G protein-coupled receptor. Ligand affinity, also known as attraction can be defined as the rate of binding agonist peptide and the G protein-coupled-receptor. It is also determined by ligand/ binding efficacy. Measurement of ligand efficacy has recently become very important in drug discovery and basic biology (Williams and Sewing, 2005). Agonists with positive efficacy, inverse agonists with negative efficacy and neutral antagonists with neutral efficacy all have different effects on the receptor and its associated signaling systems and functions.
3. Ligand efficacy and binding affinity, as illustrated above are the two requirements for demonstrating and determining specific binding and functional effects of G protein-coupled receptors. Accuracy in the measurement of these two aspects is critical in biology and determining cell activity towards agonists. Binding affinity (the strength of a protein or peptide to bind to its partner, e.g., a drug or inhibitor, is usually measured and reported by the equilibrium dissociation constant (KD). This is used to determine the order of strengths of different bimolecular interactions. The lower the KD value, the higher the binding affinity of the ligand for its target, and vice versa. There are other ways to measure binding affinities such as ELISA’s, gel-shift assays, pull-down assays, equilibrium dialysis, analytical ultracentrifugation, SPR, and spectroscopic assays. Ligand efficacy is the ability of a ligand to activate its receptor. One method that can be used to measure effectiveness directly, at the level of the receptor, is through the development of a Forster resonance energy transfer (FRET). This method allows the linkage of receptor polymorphisms with the ligand efficacy.

3.

GPCR Pathway- The ligand binds to the GPCR, which causes a conformation change in the receptor, allowing it to link to the alpha subunit of its G protein directly. This binding results in the loss of GDP and subsequent binding of GTP and also a dissociation from the G beta-gamma dimer. Often, the alpha subunit will go on to activate the next protein in the signaling pathway (which varies depending on the specific GPCR and cell type) and then give up its GTP, bind back with the G beta-gamma dimer and become inactive. In some cases, the alpha subunit is activated and separated from the GβY dimer, and this goes on to activate other proteins. This pathway is usually regulated by the end-product, and GRKs (kinases that phosphorylate the GPCR for arrestin to bind). This interrupts communication between the GPCR and the G-protein. Arrestin also binds clathrin, which forms a wall around the entire receptor and protein to internalize the complex and stop further signaling. The MAPK pathway starts with activation of Ras (a monomeric GTPase switch protein, similar to the alpha subunit of g-proteins, and is active when bound to GTP and inactive when bound to GDP). A growth factor activates Ras, that binds to its RTK, which dimerizes and phosphorylates itself. GRB2 bind to the RTK directly and the SOS binds to GRB2 and Ras. One Sos activates Ras. Ras the recruits binds and activate Raf. Raf is activated by hydrolyzing GTP to GDP, causing it to release from Ras. The active Raf activates MEK, and MEK phosphorylate and MAPK Kinase. This kinase activates several other proteins, including RSK and MNK which leads to various of the regulation of its translation and transcription functions. Cross-talk between the two pathways is essential in the activation of intracellular and intranuclear signal transduction cascades (Khan et al. 2o13)

4. Voltage-dependent calcium in the transduction process can be experimentally demonstrated via 1. Limited proteolysis of VDCC β2a and β3, Subunits, where papain is activated for 30 min in activation buffer and added to 3mg/ml VDCC β3 or β2a in a 1:2 dilutions. The final ratio of papain to protein is 1:3000. Trypsin is added, giving a final ratio of 1:1000. Reactions are performed on ice and monitored by SDS-PAGE. Products are purified for analysis by chromatography.2. Electrophysiology where, Xenopus laevis oocytes, injection of mRNA of VDCC subunits and electrophysiological recording are first performed. The negative control group used consists of oocytes, expressing CaV1.2 subunit followed by injection of the protein buffer. The positive control group is oocytes expressing a1C and β3 subunits and then a protein buffer. The experimental group is injected with a1C and then VDCC β after two days. Protein injection is then performed similarly, and incubated for one more day. Calcium channel currents are recorded using the two-electrode voltage clamp technique.
5. Gβ Protein required for peptide agonist-stimulated increases in MAPK is demonstrated through you can overexpress GβY or alpha 2-C10 adrenergic receptors that coupe to Gi in COS-7 cells. Immunoprecipitation phosphotyrosine-containing proteins revealed a 2 to 3-fold increase in the phosphorylation of two proteins of approximately 50kDa, where agonist UK-14304 was present. These proteins were also immunoprecipitated with anti-Shc antibodies and migrated with two Shc proteins. It was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase. This implied the involvement of P13K.
6. Production of AA arises as an independent effect of the receptor on phospholipase A, which directly releases AA from intact phosphoglycerides. This occurs due to phosphorylation and inactivation of the protein receptor. AA release is independent of the generation of other second messengers, including inositol phosphates, diacylglycerols, and elevation in free intracellular calcium (Burch et al., 1998). Noradrenaline binds to noradrenergic receptors located on the cell surface. Alpha, beta and gamma blockers produce AA. GTPyS stimulated AA release. Pertussis toxin partially noradrenaline-stimulated inhibited AA release, indicating the involvement of Islet activating protein. This was also inhibited by a decrease in extracellular calcium and aristolochic acid, suggesting the involvement of phospholipase A2, MAP Kinase was also reported, thus indicating the mapk pathway.
7. IAP sensitive, sensitive G protein is involved. This is suggested by cells that were pretreated with PT inhibited and phosphorylation of JNK. IAP protein was also indicated to be involved in mediating the process of pertussis toxin stimulation of AA. AA release was also directly stimulated by GTP- binding proteins when GTPyS was injected.

Works Cited:

Fillmore, D. “It’s a GPCR world.” Modern Drug Discovery. (2004) 24-28.

Kolakowski, l, f. “GCRDb: a G-Protein coupled receptor database.” Receptors and Channels. 2(1) (1994) 1-7.

King, N. Hittinger, C, Carroll, S, B. “Evolution of key cell signaling and adhesion protein families predates animal origins.” Science. 301(5631) (2003) 361.