Go to this link, which has a journal paper for which my longtime colleague Dan Kaplan was lead author: http://www.jbc.org/content/276/47/44037.full?sid=942079f2-b12b-4166-951f-d21a088d1601 and open the pdf version. Read the Abstract, then view Fig. 1 which shows a set of deletion mutants (designated A through H) that Kaplan engineered in the cytoplasmic domain of the integral plasma membrane protein E-cadherin (epithelial cadherin). To create each deletion mutant, multiple amino acids were removed by altering the cDNA that encoded E-cadherin. Fig. 2 panel C shows results for several E-cadherin deletion mutants - D, E, & F - in experiments testing their ability to bind two other proteins: b-catenin and Ga12. Fig. 2 panel C also contains a control, which tested normal E-cadherin for binding to Ga12 (panel C, upper rectangular image) and to b-catenin (panel C, lower rectangular image). Refer to material we covered in class about the cellular roles of cadherins, and answer these five questions:
i.) Which one of Kaplan’s deletion mutants (A through H) is impaired in binding the protein Ga12? ______
ii.) Regarding the region of E-cadherin deleted in mutant D, does Figure 2 panel C indicate this region is necessary for b-catenin binding, sufficient for b-catenin binding, both of these, or neither? ___________
iii.) Find the region of amino acids in E-cadherin that are not necessary for binding b-catenin but are involved in binding Ga12. Which amino acid is most frequent in this region (full name)? _______________
iv.) In Fig. 5 of the paper, human leukemia cells completely lacking E-cadherin were engineered to contain a cDNA forced these cells to express either E-cadherin (panel B), or a mutated version of E-cadherin missing the amino acids DTDPTAPPYDSLLVFDYE (panel C). Based on the microscopic images in panels A-C, explain the importance of E-cadherin/b-catenin binding in the extracellular function of E-cadherin.
v.) Let’s suppose Fig. 5 had an additional panel in which the human leukemia cells received a cDNA encoding a mutant E-cadherin with the sequence DTDPTAPPYDSLLVFDYE not deleted but changed to RTRPTAPPYKSLLVFRYH. Sketch the microscopic image you expect to observe with these cells, and explain your reasoning. [3 pts]