A G protein γ subunit-like domain shared between RGS11 and other RGS proteins specifies binding to G _β5 subunits

Abstract

Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the ␣ subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein ␥ subunit-like (GGL) domain between its Dishevelled͞Egl-10͞Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different G ␤ subunits reveals specific interaction between RGS11 and G ␤5 . The expression of mRNA for RGS11 and G ␤5 in human tissues overlaps. The G ␤5 ͞RGS11 heterodimer acts as a GAP on G ␣o , apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for G ␣ proteins and form complexes with specific G ␤ subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways.Proteins belonging to the RGS (regulators of G protein signaling) family constitute a newly appreciated group of at least 20 mammalian gene products that act as GTPaseactivating proteins (GAPs) on the ␣ subunits of heterotrimeric, signal-transducing G proteins (1-3). As such, RGS proteins can serve as negative regulators of G proteinmediated signaling pathways by speeding the inactivation of GTP-bound G ␣ subunits. Although several members of the RGS family are relatively simple Ϸ25 kDa proteins that contain little more than a characteristic RGS domain, others include modules that impart additional functions. For example, RGS12 can associate in vitro with certain G protein-coupled receptors by virtue of an alternatively spliced PDZ (PSD-95͞ Dlg͞Z0-1) domain (4), and p115, a guanine nucleotide exchange factor for the low-molecular-weight GTPase rho, contains an RGS domain that imparts sensitivity to regulation by G protein ␣ subunits (5, 6).We describe here a novel G protein ␥ subunit-like domain (GGL; pronounced giggle) that is found in several mammalian RGS proteins (RGS6, RGS7, RGS9, and RGS11) and in EGL-10, an RGS protein of Caenorhabditis elegans. The GGL domains of RGS11 and RGS7 interact preferentially with the G protein ␤ 5 subunit, and the complex of RGS11 and ␤ 5 has GAP activity toward the G protein ␣ o subunit. MATERIALS AND METHODSGeneration of Expression Constructs. cDNAs for RGS11 and various G protein subunits were cloned from human brain or retinal mRNA, from mouse retinal mRNA, or were obtained as described (7,8); all amplified cDNAs were verified by sequencing. Human RGS7 cDNA was a kind gift of Paul F. Worley (Johns Hopkins University). cDNAs encoding G protein subunits were subcloned into the mammalian expression vector pcDNA3.1-Zeo (Invitrogen), and G ␥ and RGS protein cDNAs were subcloned in-frame with an N-terminal tandem hemagglutinin (HA)-epitope tag into a modified pcDNA3.1 vector. Recombinant baculoviruses expressing native or hexahistidine-tagged RGS11 or G ␤5 subunits were generated by using the Bac-To-Bac system by following the manufacturer's protocols (Life Technologies, Gaithersburg, MD).In Vitro Transcription and Translation. Reactions were performed using the T...

The R7 Subfamily of RGS Proteins Assists Tachyphylaxis and Acute Tolerance at μ-Opioid Receptors

“…After resolving mouse cerebral cortex, hypothalamus, and PAG by SDS-PAGE, the antibodies directed to R7 proteins recognized bands of about 55 kDa for RGS7 (Kim et al, 1999;Posner et al, 1999), 75 kDa for RGS9-2 (Granneman et al, 1998;Rahman et al, 1999;, and 50 kDa for RGS11 (Snow et al, 1998). The vehicle, ODN-RD, or ODN-RGS#M administration brought about no significant variations on the immunoreactivity associated to R7 proteins observed in saline-treated mice.…”

“…The complex RGS7/b5 has GAP activity towards Goa, but not on Gi1a or Gi2a (Posner et al, 1999;Rose et al, 2000). The GAP activities of the RGS6/b5 and RGS11/b5 complexes also appear specific for Goa-GTP (Snow et al, 1998;Posner et al, 1999). However, R7 proteins have been shown to bind to activated Goa, Gi3a, and Gza subunits in rat and mouse brain membrane fractions (Saitoh et al, 1999: Rose et al, 2000Sánchez-Blázquez et al, 2003).…”

“…The DEP, and probably the R7H, domains may be involved in membrane attachment (Axelrod et al, 1998;Sondek and Siderovski, 2001). The GGL domain binds to the Gb5 protein, but not to the other Gb subunits (Snow et al, 1998;Hepler, 1999;. Contrary to that observed for other Gb1-4 subunits, the expression of Gb5 proteins is restricted to the brain (Watson et al, 1994;Fmost are found in the plasma membrane of neural cells (Watson et al, 1996;Betty et al, 1998).…”

“…The mammalian R7 proteins are also found in the CNS (Gold et al, 1997;Snow et al, 1998; Thomas et al, 1998), where they are mostly associated with cell membranes (Rose et al, 2000;. In nervous tissues, the R7 proteins and Gb5 proteins are always found as dimers, indicating that this association is required for their GAP function on the corresponding Ga-GTP subunits (Snow et al, 1998;.…”

RGSZ1 and GAIP Regulate μ- but Not δ-Opioid Receptors in Mouse CNS: Role in Tachyphylaxis and Acute Tolerance

“…Other RGS proteins also require associated proteins to act as GAP or to regulate signaling pathways. The members of the R7 subfamily of RGS proteins and Gb5 subunits are always found as dimers in the CNS, indicating that this association is required to deactivate GaGTP subunits (Snow et al, 1998;Zhang and Simonds, 2000). These R7/b5 complexes might also function as Gbg dimers, forming receptor-regulated heterotrimers with Ga subunits (Sondek and Siderovski, 2001).…”