The activation of heterotrimeric G proteins is accomplished primarily by the guanine nucleotide exchange activity of ligand-bound G protein-coupled receptors. The existence of nonreceptor guanine nucleotide exchange factors for G proteins has also been postulated. Yeast two-hybrid screens with G␣ o and G␣ s as baits were performed to identify binding partners of these proteins. Two mammalian homologs of the Caenorhabditis elegans protein Ric-8 were identified in these screens: Ric-8A (Ric-8/synembryn) and Ric-8B. Purification and biochemical characterization of recombinant Ric-8A revealed that it is a potent guanine nucleotide exchange factor for a subset of G␣ proteins including G␣ q , G␣ i1 , and G␣ o , but not G␣ s . The mechanism of Ric-8A-mediated guanine nucleotide exchange was elucidated. Ric-8A interacts with GDP-bound G␣ proteins, stimulates release of GDP, and forms a stable nucleotide-free transition state complex with the G␣ protein; this complex dissociates upon binding of GTP to G␣.Heterotrimeric guanine nucleotide-binding regulatory proteins mediate signal transduction between many membranebound receptors and intracellular effectors (1). Traditionally, activation of heterotrimeric G proteins 1 is accomplished exclusively by the action of GPCRs, seven transmembrane-spanning proteins that typically reside in the plasma membrane. These receptors act as guanine nucleotide exchange factors (GEFs), binding the inactive GDP-bound conformation of G proteins and stimulating release of GDP from G␣. To ensure directionality of exchange, GEFs stabilize a nucleotide-free transition state of G␣ that is disrupted by binding of GTP (2, 3). This facilitates dissociation of G␣⅐GTP from the G␤␥ dimer and release of these proteins from the receptor. Dissociated G protein subunits then participate in interactions with a variety of effectors.G protein signaling is attenuated when G␣ hydrolyzes the ␥ phosphate of its bound GTP and G␣⅐GDP reassociates with ␤␥. GTPase-activating proteins (GAPs) facilitate the inactivation of many G proteins. Most of these GAPs contain a regulator of G protein signaling (RGS) domain that binds preferentially to the G␣⅐GTP transition state and accelerates GTPase activity (4, 5). More than 20 unique RGS domain-containing proteins have been discovered, and the nature of their G protein specificity and their mode of action in cells are subjects of intense interest (6, 7).Nonreceptor activators of G proteins may operate in lieu of or in conjunction with GPCRs to enhance signaling, but their physiological role is not well understood (8 -11). Activators of G protein signaling AGS1 and AGS3 were identified in a genetic screen in yeast designed to isolate expressed mammalian cDNAs that encode proteins that bypass the need for a receptor (12). AGS3 possesses G␣ guanine nucleotide dissociation inhibitor activity but may activate G proteins by liberating G␤␥ (10, 13). AGS1 encodes a Ras-like small GTPase that, when bound to GTP, possesses in vitro guanine nucleotide exchange activity for members of th...

The large class of adhesion G protein-coupled receptors (aGPCRs) bind extracellular matrix or neighboring cell-surface ligands to regulate organ and tissue development through an unknown activation mechanism. We examined aGPCR activation using two prototypical aGPCRs, GPR56 and GPR110. Active dissociation of the noncovalently bound GPR56 or GPR110 extracellular domains (ECDs) from the respective seven-transmembrane (7TM) domains relieved an inhibitory influence and permitted both receptors to activate defined G protein subtypes. After ECD displacement, the newly revealed short N-terminal stalk regions of the 7TM domains were found to be essential for G protein activation. Synthetic peptides comprising these stalks potently activated GPR56 or GPR110 in vitro or in cells, demonstrating that the stalks comprise a tethered agonist that was encrypted within the ECD. Establishment of an aGPCR activation mechanism provides a rational platform for the development of aGPCR synthetic modulators that could find clinical utility toward aGPCR-directed disease.

RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab guanine nucleotide exchange factors (GEFs). Here, we show that this region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 is also shown to stimulate Rab5 guanine nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. We conclude that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 nucleotide exchange activity of RIN1.

Summary Adhesion G-protein-coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including brain development, synaptogenesis, and myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates both oligodendrocyte and cortical development. Accordingly, human GPR56 mutations cause myelination defects and brain malformations. Here, we determined the crystal structure of the GPR56 ECR, the first structure of any complete aGPCR ECR, in complex with an inverse-agonist monobody, revealing a GPCR-Autoproteolysis-Inducing domain and a previously unidentified domain that we term Pentraxin/Laminin/neurexin/sex-hormone-binding-globulin-Like (PLL). Strikingly, PLL domain deletion caused increased signaling and characterizes a GPR56 splice variant. Finally, we show that an evolutionarily conserved residue in the PLL domain is critical for oligodendrocyte development in vivo. Thus, our results suggest that the GPR56 ECR has unique and multifaceted regulatory functions, providing novel insights into aGPCR roles in neurobiology.

In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein ␣ (G␣) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and G␣ i function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to G␣ i , thus preventing its GEF activity for G␣ i . Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased G␣ i expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.