Abstract
RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gβ subunit, Gβ5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gβ5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gβ5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners.
Highlights
APRIL 22, 2016 VOLUME 291 NUMBER 17 molecules such as biogenic amines, lipids, and peptides
regulator of G protein signaling (RGS) proteins that belong to the R7 subfamily include RGS6, RGS7, RGS9, and RGS11, which are highly expressed in neurons [20] and at much lower levels in glands and the heart [21,22,23,24]
The N-terminal DEP and DHEX domains are essential for interaction of R7 proteins with R7-binding protein (R7BP) and R9-anchoring protein (R9AP), which serve as plasma membrane anchors [42,43,44,45,46,47,48]
Summary
Mice and Expression Constructs—Generation and characterization of mice with targeted deletions in R7BP [52], GPR158 [51], and G5 genes have been described [41]. Cells were washed two times with phosphatebuffered saline (PBS) and incubated with 1% paraformaldehyde (PFA) in PBS for 30 min. Under these conditions, despite formation of methylene bridges between amino groups, some protein complexes can remain soluble [54]. Whole mouse brains were minced with a razor blade to obtain pieces of tissue of ϳ1 mm3 They were washed once with PBS and incubated with 1% PFA for 30 or 60 min. Immunoprecipitation and Western Blotting—After crosslinking, transfected cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.8, 0.1% SDS, 1% Triton X-100, 0.5% deoxycholate, 1 mM EDTA, 1 mM DTT and protease inhibitors). We used mouse monoclonal anti-FLAG M2 affinity gel (Sigma, A2220), mouse monoclonal anti-HA-agarose (Sigma, A2095), and GFP-trap (Chromotek, gtp-20)
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