Regulation of human kappa opioid receptor mediated by GEC1 protein and ligands

As a typical seven-transmembrane receptor, the kappa opioid receptor (KOPR) functions at the plasma membrane and its physiological and pharmacological effects are greatly influenced by quantity of the surface receptor. In this dissertation, I examine how ligands and receptor-binding glandular epithelial cell protein 1 (GECI) affect KOPR maturation and surface expression. The studies are mainly focused on the regulatory mechanisms along the less-documented protein biosynthesis pathway.; Hydrophobic KOPR ligands are found to act on the endoplasmic reticulum as pharmacological chaperones that have facilitatory impacts on intracellular anterograde trafficking of receptor precursors. Membrane-impermeable peptide full agonists lead to receptor down-regulation to greater extents than hydrophobic ligands because they do not act as pharmacological chaperones. Membrane-permeable non-peptide antagonists act as pharmacological chaperones to induce receptor up-regulation without causing down-regulation. Membrane-permeable non-peptide agonists have two opposing effects on cell surface receptors: to decrease the receptor by causing activation-dependent endocytosis and degradation, and to increase the receptor by acting as pharmacological chaperones. Blockade of the chaperone-like effect apparently enlarges loss of surface receptor mediated by non-peptide agonist, but completely abolishes non-peptide antagonist-induced receptor up-regulation.; My data demonstrate that among the three major endogenous peptides derived from prodynorphin, alpha-neoendorphin induces lower levels of internalization and down-regulation of the hKOPR than dynorphin A and dynorphin B. The observed differential regulation is attributed not only to the lower stability of alpha-neoendorphin than dynorphin A and dynorphin B in the serum-containing culture medium, but also to the difference in their intrinsic properties in initiating these events, although all three peptides are potent full agonists with similar receptor occupation and intrinsic efficacy for activating G protein. In addition, these dynorphin peptides induce receptor phosphorylation to similar extent, excluding a possibility that the observed discrepancy is due to their different ability to mediate KOPR phosphorylation.; GEC1 is found to up-regulate KOPR in a special time- and dose-related fashion, and to promote expression of both immature and mature KOPRs. Among the three types of opioid receptors, the facilitatory effect of GEC1 is KOPR-specific. Binding with microtubule is shown to be important for GEC1 to mediate KOPR up-regulation, implying that GEC1 influences intracellular trafficking of KOPR. Modeling results suggest that these two proteins contact directly via a hydrophobic pocket of GEC1 and a lipophilic motif of KOPR C-tail. Moreover, an intramolecular hydrophobic interaction appears to be important for GECI to assume its native conformation and thus influential to GEC1-KOPR interaction.; Overall, the present studies demonstrate surface receptor expression can be highly regulated through not only the endocytosis pathway but also the biosynthesis pathway.