A novel Galpha binding consensus sequence, termed G-protein regulatory (GPR) or GoLoco motif, has been identified in a growing number of proteins, which are thought to modulate G-protein signaling. Alternative roles of GPR proteins as nucleotide exchange factors or as GDP dissociation inhibitors for Galpha have been proposed. We investigated the modulation of the GDP/GTP exchange of Gialpha(1), Goalpha, and Gsalpha by three proteins containing GPR motifs (GPR proteins), LGN-585-642, Pcp2, and RapIGAPII-23-131, to elucidate the mechanisms of GPR protein function. The GPR proteins displayed similar patterns of interaction with Gialpha(1) with the following order of affinities: Gialpha(1)GDP >> Gialpha(1)GDPAlF(4)(-) > or = Gialpha(1)GTPgammaS. No detectable binding of the GPR proteins to Gsalpha was observed. LGN-585-642, Pcp2, and RapIGAPII-23-131 inhibited the rates of spontaneous GTPgammaS binding and blocked GDP release from Gialpha(1) and Goalpha. The inhibitory effects of the GPR proteins on Gialpha(1) were significantly more potent, indicating that Gi might be a preferred target for these modulators. Our results suggest that GPR proteins are potent GDP dissociation inhibitors for Gialpha-like Galpha subunits in vitro, and in this capacity they may inhibit GPCR/Gi protein signaling in vivo.

Signal-activated G protein-coupled receptors (GPCRs) 1 stimulate GDP/GTP exchange on the ␣ subunits of G proteins. Following the activational interaction with receptors, G␣GTP and G␤␥ are released to activate their targets, which include adenylyl cyclases, phospholipases, phosphodiesterases, and ion channels (1-3). A novel class of GTPase-activating proteins (GAPs) for G proteins termed regulators of G protein signaling (RGS) has been identified (4 -6). RGS proteins share a highly conserved RGS domain, which is responsible for the GAP function. Recently, cloning of proteins critical for glial cell development resulted in the identification of the first known Drosophila RGS protein, LOCO (7). The LOCO sequence revealed significant homology to RGS12 and RGS14 within the RGS domain and three additional regions B, C, and D (7). A yeast two-hybrid screen was carried out using G i ␣ as bait in an attempt to confirm the interaction of LOCO with G␣. Interestingly, the D region, rather than the RGS domain of LOCO, was found to bind G i ␣ (7). Sequence analysis of the D region revealed that it contained a segment of homology with four ϳ20-amino acid repeats present in the human mosaic protein, LGN.LGN has been previously identified as a G i ␣ 2 -interacting protein using a yeast two-hybrid system (8).LGN is similar to the activator of G protein signaling 3 (AGS3), which was isolated in a functional screen for receptor-independent activators of heterotrimeric G protein signaling (9). Site-directed mutagenesis and protein interaction studies with AGS3 (9) indicated that the ϳ20-amino acid repeats common to AGS3, LGN, and LOCO were responsible for binding G i ␣. The ϳ20-amino acid repeats were termed the G protein regulatory (GPR) (9) or GoLOCO motif (10). The GPR motif was also identified in Purkinje cell protein-2 (Pcp2) and Rap1GAP, which were identified as G o ␣ binding partners in yeast-two hybrid screens (11,12). These studies suggest that GPR-containing proteins, hereafter termed GPR proteins, are likely to represent a diverse family of proteins that modulate G protein signaling. At present very little is known about the mechanisms and functions of GPR proteins. The yeast pheromone response pathway is mediated by G␤␥ subunits, and its GPCR-independent activation by AGS3 suggests that it may induce release of G␤␥ from G proteins (9). Pcp2 protein was shown to stimulate GDP release from G o ␣ without affecting the k cat for GTP hydrolysis, thus raising the possibility that GPR proteins may serve as guanine nucleotide exchange factors for G proteins (11). To date, no studies on the regulation of GPCR-mediated G protein activation by GPR proteins have been reported. In this study, we examined the effects of the AGS3 GPR domain (AGS3GPR) on the intrinsic guanine nucleotide exchange of G i ␣

Chimeric cGMP phosphodiesterases (PDEs) have been constructed using components of the cGMP-binding PDE (PDE5) and cone photoreceptor phosphodiesterase (PDE6␣) in order to study structure and function of the photoreceptor enzyme. A fully functional chimeric PDE6␣/PDE5 enzyme containing the PDE6␣ noncatalytic cGMP-binding sites, and the PDE5 catalytic domain has been efficiently expressed in the baculovirus/ High Five cell system. The catalytic properties of this chimera were practically indistinguishable from those of PDE5, whereas the noncatalytic cGMP binding was similar to that of native purified PDE6␣. The inhibitory Photoreceptor phosphodiesterases (PDEs) 1 serve as effector enzymes in the G protein-mediated visual transduction cascade (1-3). During transduction of the visual signal in vertebrate photoreceptor rod and cone cells, the activated G protein (transducin) ␣ subunit stimulates PDE catalytic activity by relieving the inhibitory constraint imposed by two identical inhibitory P␥ subunits. A recently adopted classification of cyclic nucleotide PDEs recognizes seven different families based on primary sequence and regulation (4). PDEs within each of the families have 60% or more homology while similarities between different families are 40% or less. According to this nomenclature, photoreceptor rod and cone PDEs comprise the PDE6 family (4). Rod photoreceptor PDE is composed of two large homologous catalytic ␣ and ␤ subunits of nearly identical size (molecular masses of 99.2 and 98.3 kDa) and two copies of an inhibitory ␥ subunit (molecular mass 9.7 kDa) (5-8). Cone PDE is composed of two identical ␣Ј subunits (molecular masses of 98.7 kDa) (9, 10), which share Ͼ60% homology with PDE6␣ and PDE6␤ (11). An inhibitory cone P␥ subunit that is highly homologous to rod P␥ and specific for a subset of cone photoreceptors has been identified (12). Recently, a rod-specific

ABSTRACTcGMP phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells that regulates the level of the second messenger, cGMP. PDE consists of catalytic a and 13 subunits (Pa and PIS) and two inhibitory 'y subunits (Py) that block PDE activity in the dark. The major inhibitory region has been localized to the C terminus of Py. The last C-terminal residues -IleIle form an important hydrophobic domain critical for the inhibition of PDE activity. In this study, mutants of Py were designed for cross-linking experiments to identify regions on Pa and P18 subunits that bind to the Py C terminus. In one of the mutants, the cysteine at position 68 was substituted with serine, and the last four C-terminal residues of Py were replaced with a single cysteine. This mutant, Py83Cys, was labeled with photoprobe 4-(Nmaleimido) benzophenone (MBP) at the cysteine residue. The labeled Py83CysMBP mutant was a more potent inhibitor of PDE activity than the unlabeled mutant, indicating that the hydrophobic MBP probe mimics the Py hydrophobic C terminus. A specific, high-yield cross-linking of up to 709% was achieved between the Py83CysMBP and PDE catalytic subunits. Pa and the N-terminally truncated P,B (lacking 147 aa residues) cross-linked to Py83CysMBP with the same efficiency. Using mass spectrometric analysis of tryptic fragments from the cross-linked PDE, we identified the site of cross-linking to aa residues 751-763 of Pa. The corresponding region of P18, P13-749-761, also may bind to the Py C terminus. Our data suggest that Py blocks PDE activity through the binding to the catalytic site of PDE, near the NKXD motif, a consensus sequence for interaction with the guanine ring of cGMP.In retinal rod cells, the visual receptor rhodopsin is activated by the absorption of a photon that leads to GTP-GDP exchange on the retinal GTP-binding protein, transducin (Gt) and to the dissociation of the a-subunit of Gt (GtaGTP) from 13yt and rhodopsin. GtaGTP then activates the effector enzyme, cGMP phosphodiesterase (PDE), by relieving the inhibitory constraint of the PDE 'y-subunits (P-y). cGMP hydrolysis by active PDE results in closure of cGMP gated channels in the plasma membrane (for reviews, see refs.

Members of the RGS family serve as