L3 Biochemistry/Molecular & Cellular Biology Data Handling

Semester 2 Session 1
Q1 Signalling Answer the following questions relating to Scatchard plots of 125I-ABMECA radioligand binding to the human A3 adenosine receptor after treatment for 24 hours with either vehicle (Control) or the A3 adenosine receptor agonist drug NECA (Drug-Treated) (data taken from Palmer et al. Mol. Pharmacol (1997) 52:632-640).

(pmol/mg/nM)

Control Drug-treated

The equations of the straight line fits are as follows:CONTROL: y = -0.184x + 0.068 DRUG-TREATED: y = -0.225x + 0.018 i) Explain in as much detail as you can what you understand by the terms Kd and Bmax (20% of total mark) ii) From the equations for the Scatchard plots, calculate the effect of NECA treatment on the Kd value for 125I-ABMECA binding at the human A3 adenosine receptor (20% of total mark) iii) From the equations for the Scatchard plots, calculate approximate Bmax values for 125IABMECA binding in Control and Drug-Treated conditions (20% of total mark) iv) Re-express the Bmax values obtained from part iii as number of receptor molecules per cell. Assume that the number of receptor molecules in one mole = 6.023x1023 and that 0.1 mg protein is equivalent to 106 cells (20% of total mark) v) Other experiments performed on these cells have revealed that there is a receptor reserve of 50%. Explain what this means (10% of total mark) vi) The human A3 adenosine receptor couples to Gi-proteins. Given that there is a receptor reserve of 50%, use the information you have obtained from the Scatchard plots to deduce the effect of a 24 hour treatment with NECA on agonist dose-response curves for Gi activation compared with control membranes (10% of total mark)

Q2 Signalling Pathways and Epistasis Genetics is a tool that has been applied with considerable success to the study of many developmental mechanisms, most notably the regulatory relationships of genes and the encoded proteins within signalling pathways. How do we know the order of protein function within a signalling pathway? Epistasis: The masking of the phenotype of one mutation by the phenotype of a mutation in another gene. The epistatic mutant is the one whose phenotype is displayed in the double mutant. Determining the epistatic relationship of genes in a signalling pathway is a powerful logical mechanism to order their relative functions. Signalling pathways often work by one protein turning on the function of a second protein etc., in a linear pathway. In the diagram below, the arrows represent positive regulation where the activity of Protein A activates protein B function etc. In principle negative regulation is also possible, but here we just consider positive. We do not need to know how the regulation works to order the function of proteins in a linear signalling pathway, but altering the phosphorylation state is a common mechanism. Protein A Protein B Protein C Protein D Outcome

In the model organism Caenorhabditis elegans the formation of the vulva, a structure used for mating and egg laying, is regulated by the activity of the RAS pathway. Much of what we know of the order of protein function within the RAS pathway came from epistasis studies on mutants in vulval formation in C. elegans. In a set of stem cells called the vulval precursor cells, signalling via the RAS pathway controls the developmental decision to generate a vulva. If this signalling is defective because of mutation of a gene or genes encoding RAS pathway components, then either no stem cells make a vulva and the animal has a vulvaless phenotype, or too many cells make a vulva causing the animal to develop several vulvae; this phenotype is described as multivulval. Therefore function of the RAS pathway controls the number of vulval precursor cells that are activated to undergo vulval development and thus controls the formation of the correct number of vulvae during development namely one. If the pathway drawn above represented the RAS pathway in C. elegans, the Outcome would be stem cells making a vulva during development. Below are lists of single and double mutants of C. elegans RAS pathway genes and their phenotypes. Loss of function mutants (lf) remove function for example deletion of the gene. The gain of function mutant (gf) of let-60 considered here causes the encoded protein to be constitutively active it cannot be turned off. The protein encoded by the wild type version of let60 can be negatively regulated by a protein not included in this problem - be aware that in a wild type animal it can be turned off. Single mutants and phenotypes: let-60 (lf) loss of function mutant phenotype vulvaless. let-60 (gf) gain of function mutant phenotype multivulval. lin-45 (lf) phenotype vulvaless let-23 (lf) phenotype vulvaless. lin-3 (lf) phenotype vulvaless. Double mutant phenotypes: lin-3 (lf), let-60 (gf) double mutant phenotype multivulval. let-23 (lf) let-60 (gf) double mutant phenotype multivulval. lin-45 (lf) let-60 (gf) double mutant phenotype vulvaless. let-60 (lf) plus any of the other (lf) alleles - phenotype vulvaless.

Questions To answer these questions, think about the single and double mutant phenotypes described above. 1) In those vulval precursor cells in wild type C. elegans that are activated to form a vulva, is the protein product of let-60 active (function turned on), or inactive? Include your logical reasoning. 2) Which gene(s) encode a protein that acts downstream of let-60? Explain your logic. 3) Which gene(s) encode a protein that acts upsteam of let-60? 4) Draw a logical linear pathway of function (like the signalling pathway drawn above) for the proteins encoded by the C. elegans genes described here. Are there any genes/proteins that you cannot order in relation to one another? If so, explain why you cannot order their relative function. Consider the following information. The lin-3 gene is NOT expressed in the vulval precursor cells, but rather in a set of adjacent cells. The let-23 gene function is required within those vulval precursor cells that go on to form a vulva. 5) From what you know about signalling and signal pathways, and using the extra information now given about lin-3 and let-23, can you refine the order of your pathway? Include an explanation of your logic. 6) If you were told that the first mutant involved in vulval formation identified was let-23, what experiment might be used to identify other genes in the pathway?