Fbxo45 inhibits calcium-sensitive proteolysis of N-cadherin and promotes neuronal differentiation.

Fbxo45 is an atypical E3 ubiquitin ligase, which specifically targets proteins for ubiquitin-mediated degradation. Fbxo45 ablation results in defective neuronal differentiation and abnormal formation of neural connections; however, the mechanisms underlying these defects are poorly understood. Using an unbiased mass spectrometry-based proteomic screen, we show here that N-cadherin is a novel interactor of Fbxo45. N-cadherin specifically interacts with Fbxo45 through two consensus motifs overlapping the site of calcium-binding and dimerization of the cadherin molecule. N-cadherin interaction with Fbxo45 is significantly abrogated by calcium treatment. Surprisingly, Fbxo45 depletion by RNAi-mediated silencing results in enhanced proteolysis of N-cadherin. Conversely, ectopic expression of Fbxo45 results in decreased proteolysis of N-cadherin. Fbxo45 depletion results in dramatic reduction in N-cadherin expression, impaired neuronal differentiation, and diminished formation of neuronal processes. Our studies reveal an unanticipated role for an F-box protein that inhibits proteolysis in the regulation of a critical biological process.

Development of a robust GABA(B) calcium signaling cell line using beta-lactamase technology and sorting.

The GABA(B) receptor is a member of the "family 3" G protein coupled receptors. The GABA(B) receptors modulate activity inwardly rectifying potassium channels and high voltage activated calcium channels. The GABA(B) receptors require heterodimerization between two subunits, GABA(B1) and GABA(B2), for functional expression. A robust functional calcium cell line was developed that contained both the human truncated GABA(B(1b)) and human truncated GABA(B(2)) receptors. The cell line was analyzed and sorted using beta-lactamase as a reporter. Single cell clones were sorted and isolated using flow cytometry based on high beta-lactamase expression. The single cell clones were further tested in a 384-well calcium mobilization assay using the Fluo-4 AM calcium indicator on the fluorescent imaging plate reader system (FLIPR). Twenty-seven clones were grown up from single cell collections and 10 clones demonstrated a high response to GABA stimulation. The 10 clones were re-evaluated based on agonist dose response and EC(50). Clone-16 was identified and utilized in high throughput screening (HTS) assay development. Using sorting and beta-lactamase as a reporter, we were able to develop a robust, functional cell-based, GABA(B), calcium mobilization assay. The cell line described here can be used for high throughput FLIPR screening and also to compare and rank the potency and selectivity of agonists, antagonists and potentiators of the GABA(B) receptor.