Differential Coupling of μ‐, δ‐, and κ‐Opioid Receptors to Gα₁₆‐Mediated Stimulation of Phospholipase C

Abstract

The~i-opioidreceptor has recently been shown to stimulate phosphoinositide-specific phospholipase C via the pertussis toxin-sensitive G15 protein. Given the promiscuous nature of G16 and the high degree of resemblance of signaling properties of the three opioid receptors, both 6-and K-opioid receptors are likely to activate G16. Interactions of 6-and K-opioid receptors with G15 were examined by coexpressing the oploid receptors and Ga15 in COS-7 cells. The 6-selective agonist [D-Pen 2,DPen5] enkephalin potently stimulated the formation of mositol phosphates in cells coexpressing the 6-opioid receptorand Ga 16. The 6-opioid receptor-mediated stimulation of phospholipase C was absolutely dependent on the coexpression of Ga16 and exhibited appropriate ligand selectivity and dose dependency. Similar transfection studies revealed only weak stimulation by the pi-opioid receptor, whereas the K-opioid receptor produced moderate phospholipase C activity. Ga16 thus appeared to interact differentially with the three opioid receptors. Radioligand binding assays indicate that the 1i-opioid receptor was expressed at a lower level than those of the 6-and K-Opioid receptors. To examine if differential coupling to Ga16 is prevalent, a panel of G5-or G1-coupled receptors was coexpressed with Ga16 in COS-7 cells and assayed for agonist-induced stimulation of phospholipase C. Activation of a2-and /12-adrenergic, dopamine D1 and D2, adenosine A1, somatostatin-l and -2, C5a, formyl peptide, and luteinizing hormone receptors all resulted in stimulation of phospholipase C, with maximal stimulations ranging from 1 .5-to almost 17-fold. These findings suggest that the promiscuous Ga16 can in fact discriminate among different receptors and that such preferential interaction might in part be due to the abundance of receptors.

G16-mediated Activation of Nuclear Factor κB by the Adenosine A1 Receptor Involves c-Src, Protein Kinase C, and ERK Signaling

“…A1R has previously been demonstrated to interact with both G␣ i (20) and G␣ 16 (21,22). Having a hematopoietic lineage, the Reh cells are known to express G␣ 16 (26).…”

“…These data implied that PTX-insensitive G proteins, which are absent in HEK 293 cells might be responsible for the CHA effects observed in Reh cells. Indeed, three such PTX-insensitive G proteins (G␣ z , G␣ 14 , and G␣ 16 ) have previously been shown to functionally interact with A1R upon co-expression with the receptor (20,21,28). Hence, G␣ z , G␣ 14 , and G␣ 16 were transiently co-expressed with A1R in HEK 293-NFB cells.…”

“…The PTX insensitivity of CHA-induced NFB activation in Reh cells signifies the involvement of G q or G 12 family members. As G␣ 16 is present in the Reh cells (26) and is known to interact with A1R (21,22), while neither G␣ q/11 nor G␣ 13 has been reported to associate with A1R, the activation signal for NFB is presumably transmitted via G 16 . Mechanistically, both cell types appear to employ a complicated network of intermediates for signal propagation.…”

“…Given the central role of NFB in mediating inflammatory and immune responses, it is reasonable to predict that A1R can regulate the activity of NFB. Signals arising from A1R can be channeled via PTX-sensitive G i (20) and PTX-insensitive G 16 (21,22) proteins, and both pathways can potentially lead to the activation of NFB. The fMLP receptor can employ both G i - (23) and G 16 -dependent (24) mechanisms to activate NFB, but the ability of A1R to activate NFB has not been reported.…”

Ligand stabilization of CXCR4/δ‐opioid receptor heterodimers reveals a mechanism for immune response regulation

“…Synthetic ligands such as [D-Pen2, D-Pen5]enkephalin (DPDPE), which have greater affinity and selectivity for the DOR, were thus generated and are used in pharmacological studies [25]. After ligand binding, DOR activates Ga i , which induces adenylate cyclase inhibition, inhibits voltage-sensitive Ca 2+ channels, activates PKC, promotes Ca 2+ release from internal stores, recruits membrane proteins such as Ras-G protein regulatory factor, and activates the ERK/MAPK transduction cascade [26][27][28][29].We show that simultaneous stimulation of CXCR4-and DOR-expressing cells by their respective ligands, CXCL12 and DPDPE, does not trigger function. We excluded differences in cell surface receptor expression and receptor internalization, as well as heterologous desensitization processes, as the cause of this effect.…”