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Morphine signaling via the μ-opioid receptor (MOR) is coupled to redox-dependent zinc release from endogenous stores. Thus, MOR activation stimulates the complex formed by RGSZ2 (a regulator of G protein signaling) and neural nitric oxide synthase (nNOS) to produce NO, and to recruit PKCγ and Raf-1 in a zinc-dependent manner. Accordingly, we investigated whether redox regulation of zinc metabolism was unique to the MOR, or if it is a signaling mechanism shared by G-protein coupled receptors (GPCRs). A physical interaction with the RGSZ2-nNOS complex was detected for the following GPCRs: neuropeptides, MOR and δ-opioid (DOR); biogenic amines, 5HT1A, 5HT2A, α2A, D1 and D2; acetylcholine, muscarinic M2 and M4; excitatory amino acid glutamate, mGlu2 and mGlu5; and derivatives of arachidonic acid (anandamide), CB1. Agonist activation of these receptors induced the release of zinc ions from the RGSZ2 zinc finger via a nNOS/NO-dependent mechanism, recruiting PKCγ and Raf-1 to the C terminus or the third internal loop of the GPCR. A series of GPCRs share an unexpected mechanistic feature, the nNOS/NO-dependent regulation of zinc ion signaling via a redox mechanism. The RGSZ2 protein emerges as a potential redox zinc switch that converts NO signals into zinc signals, thereby able to modulate the function of redox sensor proteins like PKCγ or Raf-1. Redox mechanisms are crucial for the successful propagation of GPCR signals in neurons. Thus, dysfunctions of GPCR-regulated NO/zinc signaling may contribute to neurodegenerative and mood disorders such as Alzheimer's disease and depression. Show less

Morphine increases the production of nitric oxide (NO) via the phosphoinositide 3-kinase/Akt/neural nitric oxide synthase (nNOS) pathway. Subsequently, NO enhances N-methyl-D-aspartate receptor (NMDAR)/calmodulin-dependent protein kinase II (CaMKII) cascade, diminishing the strength of morphine-activated Mu-opioid receptor (MOR) signaling. During this process, NO signaling is restricted by the association of nNOS to the MOR. Here, we examined how nNOS/NO signaling is downregulated by the morphine-activated MOR and how this regulation affects antinociception. Accordingly, we show that the MOR-NMDAR regulatory loop relies on the negative control of nNOS activity exerted by RGSZ2, a protein physically coupled to the MOR. This regulation requires binding of the nNOS N terminal PDZ domain to the RGSZ2 PDZ binding motifs that lie upstream of the RGS box. Indeed, in RGSZ2-deficient mice morphine over-stimulates the nNOS/NO/NMDAR/CaMKII pathway, causing analgesic tolerance to develop rapidly. Recovery of RGSZ2 levels or inhibition of nNOS, protein kinase C, NMDAR, or CaMKII function restores MOR signaling and morphine recovers its full analgesic potency. This RGSZ2-dependent regulation of NMDAR activity is relevant to persistent pain disorders associated with heightened NMDAR-mediated glutamate responses and the reduced antinociceptive capacity of opioids. Show less