Kappa opioid receptor (KOR), one of the three opioid receptors, is a G protein-coupled receptor (GPCR). KOR agonists produce analgesic and anti-pruritic effects, but their development for clinical use has been limited by side effects, most importantly dysphoric and psychotomimetic effects. KOR activation results in G protein- and -arrestins-dependent signaling as well as -arrestins-mediated KOR desensitization and internalization. It is thought that biased KOR agonists preferentially activating G protein-dependent pathways may produce analgesic and anti-pruritic effects with fewer side effects. For Specific Aim 3.1 of the parent funded award, we have been examining the roles of agonist-promoted phosphorylation in KOR-mediated behavioral responses by generating and characterizing mice harboring S356A/T357A/T363A/S369A mutations in the KOR (K4A). K4A mice do not undergo agonist-induced receptor phosphorylation and thus do not recruit -arrestins. For comparison, we examined KOR-mediated behaviors in -arrestin2 (-arr2) knockout (KO) mice. We found that -arr2 deletion in mice enhanced anti-scratch effects produced by the selective KOR agonist U50,488H in males, but not in females. In contrast, -arr2 deletion did not affect U50,488H-induced aversion in either male or female mice. Thus, whether -arr2 deletion has sex-specific effects depends on the behavior examined. To our knowledge, this is the first demonstration that -arr2 deletion has different effects in females vs. males on GPCR-mediated behaviors. Published studies on -arr2 KO mice have been performed in males or a mixed population of males and females. Thus, studies on female -arr2 KO mice to define possible sex differences are warranted. For this administrative supplement, we propose the following two specific aims. For specific aim 1, we will examine if there are sex differences in the effect of -arr2 deletion on the KOR-mediated analgesic effect, locomotor activity and tolerance. Experiments will be performed using U50,488H in male and female WT and -arr2 KO mice. The acetic acid writhing test will be used to assess the analgesic effect and inhibition of novelty-induced locomotor activity will be examined. Tolerance will be investigated in the anti-pruritic and analgesic tests following repeated U50,488H treatment. For specific aim 2, we will investigate if sex hormones play a role in the sex differences in effects of -arr2 deletion on U50,488H-induced behaviors. We will examine if ovariectomy (OVX) and orchidectomy (ORX) changes U50,488H-induced behavioral phenotypes in -arr2 KO mice. If so, in the future whether estrogen replacement in OVX mice and testosterone replacement in ORX mice restore the phenotypes will be examined. This is the first comprehensive investigation on the roles of - arr2 in GPCR-mediated behaviors in both males and females. Different roles of -arr2 in males vs. females in KOR-mediated behaviors have clinical implications in that -arr2 signaling-media...