Assessing Allosteric Modulation of CB₁ at the Receptor and Cellular Levels

The cannabinoid CB₁ receptor is abundant in the central nervous system and regulates neuronal transmission and other key physiological processes including those leading to pain, inflammation, memory, and feeding behavior. CB₁ is activated by the endogenous ligands, arachidonoyl ethanolamine and 2-arachidonoyl glycerol, by various synthetic ligands (e.g., CP55940), and by Δ⁹-tetrahydrocannabinol, the psychoactive component of Cannabis sativa. These CB₁ ligands are orthosteric and transduce downstream signals by binding CB₁ and primarily inducing G_i coupling, but G_s and β-arrestin coupling are also possible. Recently, allosteric modulators for CB₁ were discovered that bind to topographically distinct sites and can noncompetitively impact the potency and efficacy of orthosteric compounds. These offer the exciting potential for mechanistic analyses and for developing therapeutics. Yet, it is critical to elucidate whether a compound is a positive allosteric modulator or a negative allosteric modulator of orthosteric ligand-induced CB₁ profiles to understand pathway specificity and ameliorate diseases. In this chapter, we present equilibrium and kinetic binding analysis to reveal the impact of allosteric modulators on CB₁. Also described are activities consistent with CB₁ activation (or inactivation) and include cellular internalization of CB₁ and downstream signaling patterns. Since many CB₁ allosteric modulators do not enhance G protein coupling, it is critical to distinguish CB₁ activation and biased signaling patterns via β-arrestin from CB₁ inactivation. These strategies can illuminate pathway specificity and are valuable for the fine-tuning of CB₁ function.