Real‐time evaluation of human secretin receptor activity using cytosensor microphysiometry

Abstract

Human secretin receptor is a G protein-coupled receptor that is functionally linked to the cAMP second messenger system by stimulation of adenylate cyclase. To functionally characterize the receptor and evaluate its signal transduction pathway, the full-length human secretin receptor cDNA was subcloned into the mammalian expression vector pRc/CMV and expressed in cultured CHO cells. Intracellular cAMP accumulation of the stably transfected cells was measured by a radioimmunoassay (RIA), while the extracellular acidification rate was measured by the Cytosensor microphysiometer. Human secretin and biotinylated human secretin were equipotent in both assays in a dose-dependent manner. The EC50 values of stimulating the intracellular cAMP accumulation and the extracellular acidification rate were 0.2-0.5 nM and 0.1 nM, respectively, indicating that microphysiometry is more sensitive than the cAMP assay in monitoring ligand stimulation of the human secretin receptor. The secretin-stimulated response could be mimicked by forskolin and augmented by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, indicating that the extracellular acidification response is positively correlated with intracellular cAMP level. The response could be abolished by the protein kinase A inhibitor H-89, suggesting that protein kinase A plays an essential role in the intracellular signaling of the receptor. Upon repeated stimulation by the ligand, the peak acidification responses did not change significantly at both physiological (0.03 nM and 3 nM) and pharmacological (0.3 microM) concentrations of human secretin, suggesting that the human secretin receptor did not exhibit robust homologous desensitization.

MOLECULAR EVOLUTION OF GPCRS: Secretin/secretin receptors

“…When stimulated with secretin, all the characterized secretin receptors demonstrate a preferential downstream stimulation toward the cAMP to the intracellular calcium pathway (Siu et al 2006), with the exception of teleost secretin receptors because no endogenous ligand has been identified (Ng et al 1999, Tam et al 2011, Wang et al 2012. This suggests that the G-protein (Gas) binding domain is well conserved in receptor evolution.…”

“…6), only Asn88 is conserved in all secretin receptors. However, mutation of this residue in human SCTR did not have significant impact on the signaling and trafficking of the receptor (Pang et al 1999). High variation was found for residue Asn82.…”

Cytosensor techniques for examining signal transduction of neurohormones

“…Furthermore, this desensitisation was homologous and profound, lasting more then 12 h (Hammond et al 1999). In general, endocrine receptors display slower desensitisation kinetics than neuronal receptors (Smart et al 1998a;Ng et al 1999;Richards et al 1997), at least in microphysiometry studies. However, there are exceptions to this, e.g., the PTH receptor-induced ECAR in osteoblasts undergoes rapid desensitisation (Barrett et al 1997).…”

“…The regulation of adenylate cyclase (AC) activity and subsequent downstream events by various hormones and neuropeptides has been studied using microphysiometry (Barrett et al 1997;Ng et al 1999). Owing to the limitations of the compounds available, most studies have examined the effects of various combinations of six basic biochemical manipulations on the ECAR induced by the appropriate agonist.…”

“…Cholera-toxin treatment abolished the CRF 1 receptor-mediated ECAR in a recombinant CHO cell line , confirming the findings of a cAMP assay (Barstye et al 1999). Another study used two further approaches to examine the role of AC in the secretin-induced ECAR in recombinant cell line (Ng et al 1999). Firstly, forskolin, a turpentine that directly activates AC (Hirst and Lambert 1995), was shown to induce an ECAR in secretin receptor expressing cells, with a profile that was indistinguishable from that of the secretin-induced response (Ng et al 1999).…”