Uncovering the cellular pathways involved in Lowe syndrome
Lowe syndrome is an X-linked recessive disorder characterized by congenital cataracts, mental retardation, and abnormal kidney functions. Loss of function mutations in the OCRL1 gene lead to Lowe syndrome in all patients to date. The ocrll protein was previously identified as a PIP2 5-phosphatase localized to the trans-Golgi network. However, the mechanism through which loss of ocrll leads to the Lowe syndrome phenotype is still unknown. The aim of these studies was to define the cellular role of the PIP2 5-phosphatase, OCRL1, and its paralog, INPP5B. The first goal of this project was to study the ocrll paralog, INPP5B, in order to understand why Inpp5b can compensate for ocrll-deficiency in mice while the human ortholog cannot. In these studies, human INPP5B was found to express two distinct mRNA transcripts. The canonical transcript diverges from the murine Inpp5b transcript in the region corresponding to exon 7. This human INPP5B transcript lacks an 80-bp domain present in the murine transcript. However, a novel second alternatively spliced form of human INPP5B was found to contain this 80bp region which increases its similarity to the murine form of Inpp5b. Murine tissues express only one isoform of Inpp5b. Interestingly, in human tissues, the alternatively spliced human transcript was expressed the least in the tissues affected in Lowe syndrome patients. These results suggest that the 80bp domain present in the murine INPP5B may be responsible for the compensation of ocrll-defiency in mice. Additionally, the alternatively spliced form of human INPP5B may explain the tissue-specificity of the Lowe syndrome phenotype. These data are important for designing a mouse model for the study of Lowe syndrome pathogenesis and for the understanding of ocrll function in human tissues. The second part of the studies focused on identifying interacting protein partners of ocrll with the goal of distinguishing pathways that are affected in Lowe syndrome. These studies were done using a yeast 2-hybrid technique in conjunction with mutation analyses. Ocrll was found to interact with proteins involved in vesicle trafficking from the trans-Golgi network. The four proteins identified in the studies, a-actinin, (3COP, SCAMP2, and septin8, are each involved in different processes of vesicle formation. Using patient missense mutations, the binding sites of these proteins on the ocrll protein were mapped. The PIP2 5-phsophatase domain and the C-terminal domain were both found to be essential for protein interactions. The protein interaction results suggest that ocrll is involved in regulating vesicle formation and protein trafficking at the trans-Golgi network. The last portion of this project focused on the conserved functional domains of ocrll, in particular the C-terminal RhoGAP domain. This domain is evolutionarily conserved in both ocrll and Inpp5b but its function is not well understood. In vitro GAP assays showed that ocrll had no RhoA-, racl, or cdc42-GAP activity. Additionally, no interaction was detected between ocrll and racl or cdc42. The results suggest that the C-terminal domain does not function as a GTPase activating domain but instead functions as a binding site for protein interactions. These studies have given insight into the function of ocrll and its paralog, Inpp5b. The results from the first aim showing a distinct domain in the murine INPPSB transcript as well as the alternatively spliced form of human INPPSB suggests that this domain is involved in the overlapping functions between ocrll and Inpp5b. Future studies of these protein products will reveal the functional consequences of this domain. The second and third aim of this study identified a potential role of ocrll in Golgi trafficking and the critical domains of ocrll involved in these interactions. Although more studies are necessary to fully dissect the role of ocrll, these studies have identified pathways and protein targets that may be misregulated in Lowe syndrome.