In order to effectively generate an optically sensitive MOPR-based receptor chimera that would have high probability of structural similarity to mu-opioid receptors and retain native opioid Gi/o-protein signaling, we used the closest class A-related receptor as a backbone. Rat rhodopsin RO4 has been previously shown to couple to Gi/o signaling pathways in vitro and thus served as the ideal template for generation of an opto-MOR (). Using the constraint-based multiple alignment tool (COBALT, NCBI; http://www.st-va.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi ), rat rhodopsin RO4 was aligned against rat MOPR to identify and align transmembrane, intracellular, and extracellular domains ( Figures 1 A and S1 A). To confer MOPR-like coupling, we isolated the rhodopsin RO4 sequence, retained the critical photoisomerizing RO4 retinal binding site ( Figures S1 A and S1B), and inserted the rat MOPR intracellular loops and C terminus ( Figures S1 A and S1C). A protein folding prediction was modeled by bioinformatics software () in order to accurately project how our various chimeras would result in a seven transmembrane protein with matched features ( Figures 1 A and S1 D). This approach allowed for optimal conservation of Gi/o receptor dynamics, a high degree of photosensitivity, while simultaneously providing the critical intracellular communication components for engaging mu-opioid signaling pathways in vitro and in vivo.

(K and L) Quantification of light-induced pERK in opto-MOR (n = 6) (K) and DAMGO-induced pERK in MOPR (n = 2) (L). Data are represented as mean ± SEM. See also Figures S1 and S2

Opto-MOR Is Photosensitive and Effectively Mimics Mu-Opioid Signaling Dynamics

Bruchas et al., 2011 Bruchas M.R.

Schindler A.G.

Shankar H.

Messinger D.I.

Miyatake M.

Land B.B.

Lemos J.C.

Hagan C.E.

Neumaier J.F.

Quintana A.

et al. Selective p38α MAPK deletion in serotonergic neurons produces stress resilience in models of depression and addiction. Al-Hasani and Bruchas, 2011 Al-Hasani R.

Bruchas M.R. Molecular mechanisms of opioid receptor-dependent signaling and behavior. Belcheva et al., 2005 Belcheva M.M.

Clark A.L.

Haas P.D.

Serna J.S.

Hahn J.W.

Kiss A.

Coscia C.J. Mu and kappa opioid receptors activate ERK/MAPK via different protein kinase C isoforms and secondary messengers in astrocytes. Agonist stimulation of all four opioid receptors has been shown to recruit various factors resulting in mitogen-activated protein kinase (MAPK) activation (). MOPR has been shown to elicit a rapid initial peak in the phosphorylation of extracellular signaling-regulated kinase (pERK) in neurons, astrocytes, and transfected cell cultures (). Here, we examined whether opto-MOR and MOPR produce similar kinetics and efficacy in engaging pERK signaling in HEK293 cells. In complementary experiments, we found a rapid and transient increase in pERK (∼2–5 min) in response to blue LED photostimulation of opto-MOR and DAMGO application to MOPR-expressing cells ( Figures 1 J–1L). pERK returned to basal levels 60–90 min after either photostimulation or DAMGO treatment. Furthermore, opto-MOR-mediated activation of ERK was mostly independent of LED pulse time ( Figure S2 H) and only mildly affected by light power ( Figure S2 I), suggesting that time-locked photoactivation of opto-MOR immediately engages the MAPK signaling cascade.

in ), with a peak effect at 30 min after agonist or light stimulation ( Al-Hasani and Bruchas, 2011 Al-Hasani R.

Bruchas M.R. Molecular mechanisms of opioid receptor-dependent signaling and behavior. Figure 2 Opto-MOR and MOPR Internalization and Recovery from Desensitization Show full caption (A) Representative images show internalization of opto-MOR (colorized yellow; scale bar, 50 μm) and MOPR (inset, gray; scale bar, 10 μm) expressed in HEK293 cells in response to light and DAMGO. 0, 5, 15, 30, and 45 min time points are represented. Arrowheads show examples of internalized receptor. (B) Quantification of receptor internalization in opto-MOR (purple; τ in = 8.9 min; n = 16–43 cells per time point over 2 experimental replicates) and MOPR (gray; τ in = 8.5 min; n = 24-38 cells per time point over 3 experimental replicates) in response to light and DAMGO-induced activation, respectively. (C) Opto-MOR inhibits forskolin-induced (dashed line) cAMP following light stimulation (n = 4 traces). (D) A second light pulse 15 min following the first shows a loss of opto-MOR activity (n = 3–4 traces). (E) cAMP inhibitory activity returns to baseline levels 60 min following an initial light pulse (n = 3 traces). ∗∗∗p < 0.001 via one way ANOVA followed by Dunnett’s multiple comparison test to control). Data are represented as mean ± SEM. See also (F) Time course of recovery from desensitization (n = 3–11 replicates;p < 0.001 via one way ANOVA followed by Dunnett’s multiple comparison test to control). Data are represented as mean ± SEM. See also Figure S2 Opioid receptors are well known to be rapidly regulated by arrestin-clathrin-mediated internalization pathways. To assess whether opto-MOR exhibits similar activation-induced receptor regulation and engages canonical mu-opioid receptor internalization machinery, we performed side-by-side experiments whereby we compared the kinetics and efficacy of LED or agonist-stimulated receptor internalization on opto-MOR or MOPR. Following photostimulation or DAMGO treatment, respectively, both opto-MOR and MOPR internalized rapidly (within 15 min) with similar rates of internalization (τ), with a peak effect at 30 min after agonist or light stimulation ( Figures 2 A, 2B, S3 A, and S3B). Opto-MOR receptors remained punctate in the cytosol at all post-stimulation times tested, lasting up to an hour. Opto-MOR internalization over this time course is consistent with previously reported wild-type MOPR internalization by both widely used synthetic and endogenous ligands ().