دانلود رایگان مقاله سیگنال دهی گیرنده آدنوزین درونی در رده سلولی لیمفوبلاست

abstract

Genetic differences between individuals that affect drug action form a challenge in drug therapy. Many drugs target G protein-coupled receptors (GPCRs), and a number of receptor variants have been noted to impact drug efficacy. This, however, has never been addressed in a systematic way, and, hence, we studied real-life genetic variation of receptor function in personalized cell lines. As a showcase we studied adenosine A2A receptor (A2AR) signaling in lymphoblastoid cell lines (LCLs) derived from a family of four from the Netherlands Twin Register (NTR), using a non-invasive label-free cellular assay. The potency of a partial agonist differed significantly for one individual. Genotype comparison revealed differences in two intron SNPs including rs2236624, which has been associated with caffeine-induced sleep disorders. While further validation is needed to confirm genotype-specific effects, this set-up clearly demonstrated that LCLs are a suitable model system to study genetic influences on A2AR response in particular and GPCR responses in general.

4. Discussion

It is well established that label-free technologies can be applied to investigate GPCR signaling in heterologous as well primary adherent cell systems [23,24,33]. For instance, the xCELLigence system has successfully been applied to study ligand effects on the cannabinoid receptor 2 (CB2) and the metabotropic glutamate receptor 1 (mGluR1) using recombinant Chinese hamster ovary (CHO) cells [37]. Similarly, A2AR signaling has been studied in HEK293hA2AAR cells using selective agonists as well as partial agonists [33]. While only such recombinant cell lines have been used to study A2AR signaling using label-free technology, A2AR function has been studied in some endogenous cell types using other, more traditional assays [38–40]. However, studying a person’s A2AR response using a personal cell line such as the LCLs has not been possible up until now, and is therefore a translational step further toward precision medicine. Applicability of this label-free technology to LCLs is, however, not entirely straightforward due to their suspension cell nature. Nonetheless, adherence levels after coating of the wells with fibronectin were sufficient to allow monitoring of receptor responses, as was demonstrated by testing adenosine receptor ligands (Fig. 1). Activation of A2AR receptors led to a typical increase in impedance often seen for GPCR ligands in LCLs. For instance, P2Y receptors (Ensembl family: ENSFM00760001715026) are abundantly present on many cell types, including LCLs [41,42], which has made ATP a reference agonist for testing of functional LCL responses [21]. Interestingly, both adenosine receptor agonists and ATP display the same shape of response, which was also comparable to the response to cannabinoid receptor 2 (CB2) agonists as seen in an earlier publication [21]. Herein we showed that LCL densities of 50,000 cells/well were sufficient for detection of a robust CB2 as well as P2Y receptor response [21]. In the present study seeding densities were increased to 80,000 cells/well to obtain a window sufficient for A2AR partial agonist characterization.