Structure-Function Studies of Secreted Pdz Domain-Containing Protein 2 (Spdzd2)

This dissertation, "Structure-function Studies of Secreted PDZ Domain-containing Protein 2 (sPDZD2)" by 鄭珊, Shan, Amy, Cheng, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Structure-function studies of secreted PDZ domain-containing protein 2 (sPDZD2) Submitted by Cheng Shan Amy for the degree of Master of Philosophy at The University of Hong Kong in August 2007 PDZD2 (PDZ domain-containing protein 2), also known as AIPC (Activated in Prostate Cancer), was reported to be up-regulated in prostate tumor cells and might play a role in prostate cancer development. Proteolytic cleavage of PDZD2 by caspase 3 resulted in the secretion of a 37kDa protein sPDZD2 (secreted PDZD2) which contains two PDZ domains. Previous study in this laboratory has demonstrated the endogenous expression of PDZD2 and secretion of sPDZD2 in several prostate cancer cell lines. Importantly, the sPDZD2 protein was shown to inhibit prostate cancer cell growth. Based on these findings, sPDZD2 protein is postulated to function as a novel autocrine prostate tumor suppressor. To support this hypothesis, the structure-function relationship of this autocrine signaling protein in tumor cell growth suppression was investigated in the present studies. Four different forms of recombinant sPDZD2 peptides were constructed and synthesized: full-length (wild-type) sPDZD2, N-terminal part (sPDZD2 with C-terminal part deleted starting from the PDZ5 domain), C-terminal part (sPDZD2 with N-terminal part deleted but with retention of the C-terminal PDZ5 and PDZ6 domains) and PDZ5/6-mutated sPDZD2 (sPDZD2 with 2 mutated C-terminal PDZ domains). The effects of the mutant forms of recombinant sPDZD2 protein on prostate cancer cells were examined by MTS cell proliferation and trypan blue dye exclusion assays. Full-length sPDZD2 was shown to i inhibit the proliferation of DU145 prostate cancer cells. However, neither the N-terminal sPDZD2 nor the C-terminal sPDZD2 induced any growth-inhibitory effects on DU145 cells. The anti-proliferative effect of full-length (wild-type) sPDZD2 on DU145 cells was abolished when both the PDZ domains were mutated. The results suggested that both the functional PDZ domains in the C-terminal part and the N-terminal non-PDZ domain-containing portion of the sPDZD2 protein may be the important structural elements of the protein responsible for inducing an anti-proliferative effect on DU145 cells. Furthermore, radioligand binding studies were conducted to define some basic properties of the unknown cellular receptor. Putative high-affinity, low-density sPDZD2 receptor was shown to exist in DU145 cells. The affinity (K ) and the density (B ) of the d max putative sPDZD2 receptor on DU145 cells was in the range of 27 - 33 pM and 43.2 - 57.6 fmol/mg protein respectively. Moreover, competition binding studies showed that the mutant sPDZD2 proteins blocked the full-length sPDZD2 protein from binding to its putative receptor with similar or same potencies. Interestingly, increasing concentrations of different mutant forms of sPDZD2 were also noted to attenuate and abrogate, ultimately, the anti-proliferative effect of wild-type sPDZD2 on DU145 cells. Together, the data implicate significant roles played by the functional PDZ domains in the C-terminal part and the N-terminal non-PDZ domain-containing portion of the sPDZD2 protein in receptor binding and activation, leading to prostate cancer cell anti-proliferation. Of note, the present investigation represented the first step towards understanding the signaling mec