Receptors As Basic Drug Recognition Units

In lieu of direct biochemical characterization, historically, receptor classification was based on the observed system-independent measurement of agonist potency ratios (PRs) and antagonist equilibrium dissociation constants. This approach depends on the concept that the receptor is the single discerning unit for agonism. Under these circumstances, the affinity and efficacy terms in Equation 1.9 (namely KA and t respectively) refer to the specific interaction of a given agonist for a given receptor (irrespective of which cell type response is mediated). Under these circumstances, the magnitude of the relative ratio of potency (PR) for two full agonists (A and B) is a unique identifier of the agonists and receptor type since it is independent of all tissue-based response elements:

Deviation of such PR estimates were considered to be presumptive evidence of differences in the receptor (as the minimal recognition unit). The emphasis of classical pharmacology was recognition of chemicals since the response systems usually came as an intact unit (i.e., isolated tissues). However, the independent nature of the receptor and response elements (a "floating " receptor interacting with different free G proteins and other cytosolic proteins) adds another element of recognition, namely the recognition of the response element after agonist binding. The impact of this factor became clear with the use of recombinant receptor systems where the relative stoichiometry of these elements could be varied. In addition, technological advances furnished the means to selectively observe the individual components of cellular response, thereby allowing the measurement of changes in distinct receptor-coupled pathways to agonist activation. These types of systems furnished experimental data that was totally inconsistent with the previously described assumptions concerning PRs of agonists. Specifically, it was observed that the potency of agonists for a single receptor differed when different response cascades, mediated by that same receptor, were measured. For example, for pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which pleiotropically mediate changes in cyclic adenosine monophosphate (AMP) and inositol phosphate 3 (IP3), two PACAP peptide fragments (PACAP1-27 and PACAP1-38) produced elevated cyclic AMP and IP3 in cells. However, the relative potency of these two agonists for these pathways was reversed [55]. Thus, the relative efficacy of PACAP1 - 27 for cyclic AMP elevation is higher than that for PACAP1 - 38 but lower for elevation of IP3 . In historical terms, this would have implied that the responses to the two agonists were mediated by different receptors. Since only one receptor type was transfected into the cell, this was not an option requiring consideration of alternative ideas. It should be noted that relative ratios of potency need not be reversed to denote functional selectivity since the actual numerical value of the PR accurately depends on relative affinity and efficacy of the agonist for the receptor recognition unit.

A tacit assumption in the historical view of agonist PRs is the idea that all agonists produce a uniform receptor-active state, that is, they all flip the receptor switch in an identical manner. However, there is an abundance of evidence to show that proteins spontaneously produce a myriad of conformations in response to thermal energy [56-59] . In accordance to the concept of confor-mational selection [28], ligands interact with these collections of conformations (termed "ensembles") and stabilize subsets of them through selectively high affinity [60, 61] . If there were a collection of conformations, then some may have greater affinity for some response elements than others. Under these circumstances, the minimal recognition unit, with respect to the response elements of the cell, would not be the receptor per se but rather the receptor-active state (i.e., ligand-stabilized subset of receptor conformations made after ligand binding). If the active state were the minimal recognition unit, then the experimental results indicating differing PRs for different response pathways can be accommodated. Such differences require that different ligands stabilize different ensembles of receptors to produce different macro-active states interacting with the cell.

The varying PRs observed for PACAP analogs led to a modified model of receptor stimulus whereby stimulus could "traffic" to different portions of a cellular stimulus-response cascade [62] . There has been a large body of evidence since that time to verify this mechanism with many ligands (see References 63-67 for reviews of specific papers), suggesting that many ligands produce different receptor-active states. This is, in fact, consistent with experimental observations. There is another large body of data from a number of experimental approaches that confirm that ligands can stabilize different receptor conformations [ 68-72]. This idea completes the general notion of 7TM receptors as microprocessors. Thus, a range of transient receptor conformations can be stabilized by different ligands to form varying predominant conformations that then interact with a range of independent cytosolic reac-tants to produce cellular response.

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