GPCRs binding assay

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germix's picture
GPCRs binding assay

I am working with two different cell-line (native human NK1 receptor expressed in astrocytoma UC11MG and recombinant CHO cells) to investigate the signal trasduction pathway of the NK1 receptors (GPCRs family). I used an amide series of compounds to determine the SAFIR relationships, but Ive a problem to rationalize the results. These amides are secondary and tertiary and with the first cell-line the secondary are more potent of the tertiary, using the recombinant cell-line the situation is opposite. Is that a common behavior? The IC50 values arent comparable but the form, slope, Bmax of the curves are very similar. Im sorry but pharmacology is not my field. Any help would be greatly appreciated,

kumar's picture
You might be onto something

You might be onto something interesting, take a look at this:
Mol Pharmacol. 2000 Dec;58(6):1230-8.
The use of stimulus-biased assay systems to detect agonist-specific receptor active states: implications for the trafficking of receptor stimulus by agonists.

* Watson C,* Chen G,* Irving P,* Way J,* Chen WJ,* Kenakin T.

Department of Receptor Biochemistry, Glaxo Wellcome Research and Development, Research Triangle Park, North Carolina.

The quantitative comparison of the relative potency of agonists is a standard method of receptor and agonist classification. If agonist potency ratios do not correspond in two given tissues, this is used as presumptive data to conclude that the receptors in those two tissues are different. This article presents data to show that a single receptor can demonstrate varying agonist potency ratios in different host cells. These data are described in terms of the production of more than one agonist-selective receptor active state and the interaction of these different active states with multiple G proteins in the membrane to produce cellular response. Stable host human embryonic kidney 293 cells with enhanced quantities of the respective Galpha-protein were created. Wild-type and Galpha-subunit enriched cells were then transiently transfected with human calcitonin receptor type 2 (hCTR2). Binding did not detect differences in the G protein-enriched cells versus wild-type cells. In contrast, functional studies did show differences between the host cell lines and Galpha-subunit enriched cell lines. The relative potency of eight calcitonin agonists was measured in studies of calcium fluorescence in transfected cells containing human calcitonin receptor type 2 by comparing pEC(50) (-log molar concentration producing half-maximal response) values. In Galphas-enriched cells, the relative order of potency of the agonists changed. The host-cell dependent differences in potency ratios ranged from 2-fold to more than 46-fold. This finding is not consistent with the idea that all of the agonists produce response in the same manner (i.e., through a common active state of the receptor). These data are consistent with the idea that these different agonists produce arrays of active states that differentially use G proteins. This idea is discussed in terms of the design of stimulus-bias assay systems to detect agonist-selective receptor active states with resulting potential for increased selectivity of agonists.

PMID: 11093758 [PubMed - indexed for MEDLINE]

germix's picture
thanks for the review, my

thanks for the review, my problem now is that I don't know if my compounds are agonist or not..we will see.