Identifying new players and pathways involved in receptor‐mediated heterologous sensitization of adenylyl cyclase using genome‐wide siRNA and BiFC library screening

Heterologous sensitization of adenylyl cyclase (AC) is a neuroadaptive response to chronic activation of G i/o ‐coupled receptors, and is implicated in the etiology of drug addiction, schizophrenia, and treatment of Parkinson's disease. Acute activation of inhibitory receptors results in inhibition of cAMP accumulation, whereas chronic activation results in increased cAMP levels. Although heterologous sensitization was first observed four decades ago, the underlying molecular mechanisms are only partially understood. To address this problem, we developed two unbiased approaches to unearth novel players and pathways involved in sensitization. We present here the development of a robust cell‐based assay of D 2 dopamine receptor (D2R) mediated heterologous sensitization of AC6 that is suitable for high throughput screening (HTS) applications. To identify proteins involved in any aspect of heterologous sensitization, we developed and executed a genome‐wide, siRNA‐based screen. siRNA knockdown targeting individual genes was assessed as % inhibition of the sensitization phenotype by measuring cAMP accumulation. Initial statistic and bioinformatic analyses revealed that approximately 1% of the targets fit the hit criteria for cell viability and changes in activity. The initial hits represent a diverse group of proteins and pathways. Differential regulation of the nine membrane bound AC isoforms adds to the complexity of the mechanisms of heterologous sensitization. To examine whether the individual hits and pathways identified are unique to AC6, we have developed cell‐based HTS‐amenable assays for siRNA for additional AC isoforms. We also developed a screening strategy using bimolecular fluorescence complementation (BiFC) to identify novel protein‐protein interactions in living neuronal cells. A cell model co‐expressing the D2R as well as AC5 fused to a BiFC fragment of Venus fluorescent protein was constructed. A human brain cDNA library was also developed in which the individual cDNAs were linked to the complementary BiFC fragment. Following transfection of the cDNA library, cells were treated with either vehicle or the D2R agonist quinpirole to induce sensitization. Cells that exhibited an AC5‐protein interaction, as determined by BiFC, were selected using fluorescence activated cell sorting and isolated for cDNA amplification. Next generation sequencing was used to identify the potential interacting proteins from the complementing cDNAs. By comparing BiFC interactions in cells treated with vehicle versus chronic quinpirole, we expect to identify novel AC5‐protein interactions involved with the D2R‐mediated heterologous sensitization. Further validation and comparison of the results from both approaches using bioinformatic analyses and experimental studies are presently underway to pinpoint individual proteins and pathways implicated in heterologous sensitization of AC. Support or Funding Information National Institutes of Health MH096927 and MH101673