Interaction Of RIN1 With Spinophilin In Spinal Cord Dorsal Horn And Its Regulatory Mechanisms

Objective: Spinophilin can interact with multiple signaling and scaffolding proteins such as extracellular signal-regulated kinase(ERK1/2), protein phosphatase-1(PP1)and postsynaptic density-95(PSD-95). As one of the anchoring proteins, Spinophilin can position its binding partners in specific subcellular compartments and thus,determine their functional specificity. Previous studies have demonstrated that Spinophilin is expressed in the superficial dorsal horn of spinal cord, engaged in the suppression of nociceptive synaptic transmission mediated by excitatory glutamate receptors, and involved in the alleviation of mechanical allodynia produced by peripheral tissue injury. RIN1(Ras and Rab Interacting protein-1) is a multifunctional intracellular signaling protein that plays an important role in the regulation of ERK1/2,small GTPase Rab5 and protein tyrosine kinase ABL. But the function of RIN1 is still far from clear in the spinal cord. In this study, we examined the possible interaction between Spinophilin and RIN1 and investigated its regulatory mechanisms.Methods: The current study performed co-immunoprecipitation experiments to investigate the interaction of Spinophilin with RIN1 in spinal cord dorsal horn. We injected N-methyl-D-aspartate(NMDA), metabotropic glutamate receptor 1/5(m Glu R1/5) agonist DHPG intrathecally, and injected Complete Freund’s Adjuvant(CFA) into the plantar sufaces to observe if NMDA receptor, m Glu R1/5 and peripheral inflammatory injury regulated Spinophilin association with spinal RIN1.Results:(1) Immunoblotting analysis illustrated that RIN1 is present in the spinal cord of adult rats;(2) Co-immunoprecipitation experiment showed that the specific antibody against Spinophilin was able to precipitate RIN1 from spinal dorsal horn.Reciprocally, the specific antibody against RIN1 could also co-precipitate Spinophilin,suggesting that RIN1 physically interacted with Spinophilin directly or indirectly;(3) Intraplantar injection of CFA(20μl) in adult rats significantly reduced the paw withdrawal thresholds(PWT) in response to the Von Frey filament stimulation,indicating the establishment of inflammatory mechanical allodynia. Meanwhile,anti-Spinophilin antibody precipitated less amount of RIN1 in CFA-injected rats when compared to saline-injected control littermates, suggested that peripheral tissue damage disrupted the interaction between RIN1 and Spinophilin;(4) Intrathecal injection of NMDA receptor agonist NMDA(1μg) or m Glu R1/5 agonist DHPG(20μg)in normal adult rats not only reduced the paw withdrawal thresholds but impaired the interaction of RIN1 and Spinophilin, suggesting that RIN1/Spinophilin complex was subjected to the regulation by NMDA receptor and m Glu R1/5 receptor;(5) In addition,the altered activity of protein phosphatases might also influence RIN1 association of Spinophinlin, because intrathecal injection of calcineurin inhibitor Cyclosporine A(50μg) reduced the Spinophilin content precipitated by anti-RIN1 antibody from the spinal cord dorsal horn of intact adult rats;(6) NMDA receptor NR2 A subunit might complex with RIN1 through Spinophilin.Conclusion: There was an intimate interaction between Spinophilin and RIN1 in spinal cord dorsal horn, which could be modulated by NMDA receptor、m Glu R1/5receptor and protein phosphatases.