Abstract

Spinophilin (SPL) and neurabin (NRB) are structurally similar scaffolding proteins with several protein binding modules, including actin and PP1 binding motifs and PDZ and coiled-coil domains. SPL also binds regulators of G protein signaling (RGS) proteins and the third intracellular loop (3iL) of G protein-coupled receptors (GPCRs) to reduce the intensity of Ca²⁺ signaling by GPCRs. The role of NRB in Ca²⁺ signaling is not known. In the present work, we used biochemical and functional assays in model systems and in SPL^-/- and NRB^-/- mice to show that SPL and NRB reciprocally regulate Ca²⁺ signaling by GPCRs. Thus, SPL and NRB bind all members of the R4 subfamily of RGS proteins tested (RGS1, RGS2, RGS4, RGS16) and GAIP. By contract, SPL, but not NRB, binds the 3iL of the GPCRs _1B-adrenergic (_1BAR), dopamine, CCKA, CCKB and the muscarinic M3 receptors. Coexpression of SPL or NRB with the _1BAR in Xenopus oocytes revealed that SPL reduces, whereas NRB increases, the intensity of Ca²⁺ signaling by _1BAR. Accordingly, deletion of SPL in mice enhanced binding of RGS2 to NRB and Ca²⁺ signaling by AR, whereas deletion of NRB enhanced binding of RGS2 to SPL and reduced Ca²⁺ signaling by AR. This was due to reciprocal modulation by SPL and NRB of the potency of RGS2 to inhibit Ca²⁺ signaling by AR. These findings suggest a novel mechanism of regulation of GPCR-mediated Ca²⁺ signaling in which SPL/NRB forms a functional pair of opposing regulators that modulates Ca²⁺ signaling intensity by GPCRs by determining the extent of inhibition by the R4 family of RGS proteins.