Journal of Pharmacological and Toxicological Methods
Original articlesConstitutive receptor systems for drug discovery
Introduction
With the introduction of human recombinant receptor systems has come a revolution in the science of drug discovery. Additionally, obviating the species gap by screening on human rather than animal gene products, recombinant systems enable another approach to drug screening—namely, the use of constitutive receptor activity. G-protein–coupled receptors (GPCRs) can exist in inactive (denoted Ri), with respect to activation of G proteins, and active (denoted Ra) states. The degree to which this can occur depends on the energy barrier for formation of the active state and an equilibrium defined by an allosteric constant (denoted L and defined as [Ra]/[Ri]). Constitutive activity results from the interaction of a spontaneously formed active state of the receptor with one or more G proteins to produce an elevated basal level of response.
Under physiological conditions, the equilibrium lies far to the left and most natural systems are quiescent (i.e., demonstrate no constitutive receptor activity). In these systems, endogenous hormones and transmitters are required to produce active-state receptor for physiological signaling. However, recombinant cellular systems can be made constitutively active. The most universally applied method of producing visible constitutive activity is to increase the amount of active-state receptor through manipulation of the relative stoichiometry of the receptors and G proteins. This does not change the ability of the receptor to form an active state but rather increases the number of active-state receptors in the system. For example, given a receptor with an allosteric constant of 10−3, one in every 1000 receptors exists in the active form. If the receptor expression level is 1000, this one activated receptor may not be adequate to trigger visible constitutive response. However, if the expression level were to be raised to 100,000, then 100 receptors would be in the active state, a level that could be comensurate with visible constitutive activity. High levels of receptor expression have been shown to produce constitutive activity for a number of receptors Barker et al. 1994, Samama et al. 1993, Tian et al. 1994, Van Sande et al. 1995, Whaley et al. 1994.
Section snippets
Reporter assays
LucLite luciferase reporter gene assay kit was obtained from Packard (6016911, Meriden, CT). DMEM/F12 medium (11039-021), fetal bovine serum (16140-071), L-glutamine (25030-081), 0.05% trypsin (25300-054), phosphate-buffered saline (14190-144), and lipofectamine (18324-012) were obtained from GIBCO BRL (Gaithersburg, MD). Black 96-well (3904) and clear 24-well tissue culture-treated plates (3526) were obtained from Corning Costar (Cambridge, MA). Plate seals (097-05-00006) were from
Dependence of constitutive receptor activity on receptor density
The relation between receptor density and constitutive activity can be derived from the cubic ternary complex model for GPCRs, shown in Figure 1A Weiss et al. 1996a, Weiss et al. 1996b, Weiss et al. 1996c. It should be noted that very similar expressions can be derived from the extended ternary complex model for GPCRs (Samama et al., 1993), except that higher levels of maximal constitutive receptor activity are predicted by this model (vide infra). The response as a function of receptor density
Discussion
The ternary complex model for GPCRs predicts two theoretical advantages of screening in constitutive GPCR systems—namely, an increased capability to detect ligands (i.e., the added capacity to detect inverse agonists) and an increased sensitivity to agonists. This limited study of four receptor types supports these ideas. However, as seen with the GLP-1 receptor, the ability to create constitutively active screening systems depends on the receptor type (the magnitude of L), and the approach
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