A common molecular motif characterizes extracellular allosteric enhancers of GPCR aminergic receptors and suggests enhancer mechanism of action

Abstract

Several classes of compounds that have no intrinsic activity on aminergic systems nonetheless enhance the potency of aminergic receptor ligands three-fold or more while significantly increasing their duration of activity, preventing tachyphylaxis and reversing fade. Enhancer compounds include ascorbic acid, ethylenediaminetetraacetic acid, corticosteroids, opioid peptides, opiates and opiate antagonists. This paper provides the first review of aminergic enhancement, demonstrating that all enhancers have a common, inobvious molecular motif and work through a common mechanism that is manifested by three common characteristics. First, aminergic enhancers bind directly to the amines they enhance, suggesting that the common structural motif is reflected in common binding targets. Second, one common target is the first extracellular loop of aminergic receptors. Third, at least some enhancers are antiphosphodiesterases. These observations suggest that aminergic enhancers act on the extracellular surface of aminergic receptors to keep the receptor in its high affinity state, trapping the ligand inside the receptor. Enhancer binding produces allosteric modifications of the receptor structure that interfere with phosphorylation of the receptor, thereby inhibiting down-regulation of the receptor. The mechanism explains how enhancers potentiate aminergic activity and increase duration of activity and makes testable predictions about additional compounds that should act as aminergic enhancers.

Fig. (1)

Common Motif In Enhancer Structures. All known enhancers have a pair of hydroxyls and a carbonyl in a linear array within 4-6 angstroms (400-600 pm) of each other (circled).

Fig. (2)

Overlays of Enhancers Demonstrating Shared Arrays of Hydroxyls and Carbonyls. Left: Overlay of morphine (black) and aldosterone (green). Right: Overlay of naloxone (red) and EDTA (blue).

Fig. (3)

The Common Motif Identified In Enhancers In Figure 1 Is Lacking In All Known Non-Enhancers.

Fig. (4)

Predicted Aminergic Enhancers. Based on the common motif introduced in Fig. (1); we predict that the compounds illustrated here will also enhance adrenergic compounds.

Fig. (5)

Binding Site of Enhancers. Probable binding site of enhancers (orange) in relation to aminergic agonists (red) [based on studies by Dillon, et al. (8-11)].

Fig. (6)

Model of Enhancer Inhibition of GPCR Phosphorylation. Proposed model of enhancement of aminergic GPCR based on receptor allostery. Top Row: Binding of an amine to a GPCR normally results in allosteric changes in the receptor that initiate a series of molecular signals recruiting receptor kinases (indicated by the change of the intracellular loops from white to black and back again), which phosphorylate the receptor resulting in its down regulation and internalization. Bottom Row: We propose that binding of enhancers to GPCR results in prevention of aminergic release from the receptor accompanied by interference with the allosteric signaling (intracellular loops remain metaphorically in the “black” state) that initiates receptor kinase recruitment. Retention of the amine in the binding site accompanied by retardation of receptor phosphorylation results in prevention of tachyphylaxis. We also predict that during the first steps of receptor down-regulation binding of enhancers to the receptor can induce allosteric changes in the receptor that reverse receptor phosphorylation.; thereby also reversing fade.

Fig. (7)

Reversal of Smooth Muscle Fade and Tachyphylaxis by Enhancer Molecules. The graphs show the influence of enhancers on GPCR adrenergic- or histaminergic-induced smooth muscle contractions. Agonist is shown as increasing concentration (arrow) over the yellow-red lines. Force increases are shown on the Z-axis. Enhancer concentration increases over the green-blue lines. Enhancers do not induce any contraction nor do they cause any increase in maximum force (efficacy), from which it is concluded that they do not cause calcium increases. Enhancers increase the potency by 0.5-1.0 log units, resulting in greater force at sub-maximal agonist concentrations. During prolonged contractions, force fades despite the continued presence of agonist, shown by the fall in force at zero enhancer (green shaded area) in the figures from left to right. No fade occurs at high enhancer concentrations (blue shaded area). Fade is thought to occur through phosphorylation of the GPCR receptor, shown in the red arrow. Fade can be reversed in a continuous contraction by the addition of enhancer, shown in the blue arrow in the right hand graph. Since there is no evidence that any enhancer increases intracellular calcium, the reversal of fade must reverse receptor phosphorylation. Similarly, the reversal of tachyphylaxis (loss of force in sequential activations) can also be reversed by enhancers (green arrow in the right hand graph), when both the agonist and enhancer are added simultaneously at the start of an activation that would otherwise not produce force due to tachyphylaxis. The evidence indicates that the reversals of fade and of tachyphylaxis by enhancers are due to the common mechanism of reversing phosphorylation of GPCR receptors.