Brillet Karl, Kieffer Brigitte L, Massotte Dominique
Département des Récepteurs et Protéines Membranaires, UPR 9050, Ecole Supérieure de Biotechnologie de Strasbourg, F-67400 Illkirch-Graffenstaden, France.
BMC Pharmacol. 2003 Dec 1;3:14. doi: 10.1186/1471-2210-3-14.
The concept of spontaneous- or constitutive-activity has become widely accepted and verified for numerous G protein-coupled receptors and this ligand-independent activity is also acknowledged to play a role in some pathologies. Constitutive activity has been reported for the mu opioid receptor. In some cases the increase in receptor basal activity was induced by chronic morphine administration suggesting that constitutive activity may contribute to the development of drug tolerance and dependence. Constitutively active mutants represent excellent tools for gathering information about the mechanisms of receptor activation and the possible physiological relevance of spontaneous receptor activity. The high basal level of activity of these mutants also allows for easier identification of inverse agonists, defined as ligands able to suppress spontaneous receptor activity, and leads to a better comprehension of their modulatory effects as well as possible in vivo use.
Cysteines 348 and 353 of the human mu opioid receptor (hMOR) were mutated into alanines and Ala348,353 hMOR was stably expressed in HEK 293 cells. [35S] GTPgammaS binding experiments revealed that Ala348,353 hMOR basal activity was significantly higher when compared to hMOR, suggesting that the mutant receptor is constitutively active. [35S] GTPgammaS binding was decreased by cyprodime or CTOP indicating that both ligands have inverse agonist properties. All tested agonists exhibited binding affinities higher for Ala348,353 hMOR than for hMOR, with the exception of endogenous opioid peptides. Antagonist affinity remained virtually unchanged except for CTOP and cyprodime that bound the double mutant with higher affinities. The agonists DAMGO and morphine showed enhanced potency for the Ala348,353 hMOR receptor in [35S] GTPgammaS experiments. Finally, pretreatment with the antagonists naloxone, cyprodime or CTOP significantly increased Ala348,353 hMOR expression.
Taken together our data indicate that the double C348/353A mutation results in a constitutively active conformation of hMOR that is still activated by agonists. This is the first report of a stable CAM of hMOR with the potential to screen for inverse agonists.
自发活性或组成性活性的概念已被广泛接受并在众多G蛋白偶联受体中得到验证，这种不依赖配体的活性也被认为在某些病理过程中起作用。已报道μ阿片受体存在组成性活性。在某些情况下，慢性给予吗啡可诱导受体基础活性增加，这表明组成性活性可能有助于药物耐受性和依赖性的发展。组成性活性突变体是获取有关受体激活机制及自发受体活性可能的生理相关性信息的优秀工具。这些突变体的高基础活性水平也便于更容易地鉴定反向激动剂，反向激动剂定义为能够抑制自发受体活性的配体，并有助于更好地理解其调节作用以及可能的体内应用。
将人μ阿片受体（hMOR）的半胱氨酸348和353突变为丙氨酸，Ala348,353 hMOR在HEK 293细胞中稳定表达。[35S]GTPγS结合实验表明，与hMOR相比，Ala348,353 hMOR的基础活性显著更高，表明突变受体具有组成性活性。赛庚啶或CTOP可降低[35S]GTPγS结合，表明这两种配体均具有反向激动剂特性。除内源性阿片肽外，所有测试的激动剂对Ala348,353 hMOR的结合亲和力均高于对hMOR的结合亲和力。拮抗剂亲和力基本保持不变，但CTOP和赛庚啶与双突变体的结合亲和力更高。在[35S]GTPγS实验中，激动剂DAMGO和吗啡对Ala348,353 hMOR受体的效力增强。最后，用拮抗剂纳洛酮、赛庚啶或CTOP预处理可显著增加Ala348,353 hMOR的表达。
综合我们的数据表明，双C348/353A突变导致hMOR形成组成性活性构象，该构象仍可被激动剂激活。这是关于具有筛选反向激动剂潜力的hMOR稳定组成性活性突变体的首次报道。
