Frizzled-7 turnover at the plasma membrane is regulated by cell density and the Ca(2+) -dependent protease calpain-1

Frizzled are seven-transmembrane domain G-protein coupled receptors involved in cell polarity and Wnt signaling. The mechanisms regulating their turnover at the plasma membrane remain unclear. We have identified a regulated C-terminus cleavage of Frizzled-7 in endothelial cells using ectopic expression of N- and C-termini-tagged Frizzled-7 proteins. This specific cleavage produced a 10 kDa C-terminus fragment that remained associated with intracellular vesicles and was localized within the 3rd intracytoplasmic loop using N-terminal sequencing and targeted mutagenesis. Frizzled-7 mutated forms displaying reduced C-terminus cleavage were also defective for dvl2 translocation at the plasma membrane. PMA, an activator of PKC and endocytosis, but not Wnt13A and Wnt5A, increased the appearance of Frizzled-7 C-terminus-containing vesicles and Frizzled-7 cleavage. Concanavalin-A, an inhibitor of receptor internalization decreased both constitutive and PMA-induced Frizzled-7 cleavage, while inhibition of the endocytic pathway with Delta95-295-Eps15 dominant-negative prevented only PMA-induced Frizzled-7 cleavage. Frizzled-7 C-terminus cleavage was increased with cell density and by the Ca(2+) ionophore ionomycin and was decreased by specific calpain inhibitors, by the expression of DN-calpain-1 and the down-regulation of calpain-1 levels by siRNAs. Altogether, our findings pinpoint calpain-1 as a regulator of Frizzled-7 turnover at the plasma membrane and reveal a link between Frizzled-7 cleavage and its activity.     

The third intracellular loop of D1 and D5 dopaminergic receptors dictates their subtype-specific PKC-induced sensitization and desensitization in a receptor conformation-dependent manner

We previously showed that phorbol-12-myristate-13-acetate (PMA) mediates a robust PKC-dependent sensitization and desensitization of the highly homologous human Gs protein and adenylyl cyclase (AC)-linked D1 (hD1R) and D5 (hD5R) dopaminergic receptors, respectively. Here, we demonstrate using forskolin-mediated AC stimulation that PMA-mediated hD1R sensitization and hD5R desensitization is not associated with changes in AC activity. We next employed a series of chimeric hD1R and hD5R to delineate the underlying structural determinants dictating the subtype-specific regulation of human D1-like receptors by PMA. We first used chimeric receptors in which the whole terminal region (TR) spanning from the extracellular face of transmembrane domain 6 to the end of cytoplasmic tail (CT) or CT alone were exchanged between hD1R and hD5R. CT and TR swaps lead to chimeric hD1R and hD5R retaining PMA-induced sensitization and desensitization of wild type parent receptors. In striking contrast, hD1R sensitization and hD5R desensitization mediated by PMA are correspondingly switched to PMA-induced receptor desensitization and sensitization following the IL3 swap between hD1R and hD5R. Cell treatment with the PKC blocker, Gö6983, inhibits PMA-induced regulation of these chimeric receptors in a similar fashion to wild type receptors. Further studies with chimeras constructed by exchanging IL3 and TR show that PMA-induced regulation of these chimeras remains fully switched relative to their respective wild type parent receptor. Interestingly, results obtained with the exchange of IL3 and TR also reveal that the D1-like subtype-specific regulation by PMA, while fully dictated by IL3, can be modulated in a receptor conformation-dependent manner. Overall, our results strongly suggest that IL3 is the critical determinant underlying the subtype-specific regulation of human D1-like receptor responsiveness by PKC.     

An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies and gene therapy

The various iterations of the HapMap Project and many genome-wide association studies (GWAS) have identified hundreds of potential genes involved in monogenic and multifactorial traits. We constructed an arrayed 115,000-member human genomic library in the PAC shuttle vector pJCPAC-Mam2 that can be propagated in both bacterial and human cells. The library appears to represent a two-fold coverage of the human genome. Transient transfection of a p53-containing PAC clone into p53-null Saos-2 human osteosarcoma cells demonstrated that both p53 mRNA and protein were produced. Additionally, expression of the p53 protein triggered apoptosis in a subset of the Saos-2 cells. This library should serve as a valuable resource to validate potential disease genes identified by GWAS in human cell lines and in animal models. Also, individual library members could potentially be used for gene therapy trials for a variety of recessive disorders.