Seven-transmembrane receptor (7TMR) signaling is transduced by second messengers such as diacylglycerol (DAG) generated in response to the heterotrimeric guanine nucleotide–binding protein G q and is terminated by receptor desensitization and degradation of the second messengers. We show that β-arrestins coordinate both processes for the G q -coupled M1 muscarinic receptor. β-Arrestins physically interact with diacylglycerol kinases (DGKs), enzymes that degrade DAG. Moreover, β-arrestins are essential for conversion of DAG to phosphatidic acid after agonist stimulation, and this activity requires recruitment of the β-arrestin–DGK complex to activated 7TMRs. The dual function of β-arrestins, limiting production of diacylglycerol (by receptor desensitization) while enhancing its rate of degradation, is analogous to their ability to recruit adenosine 3′,5′-monophosphate phosphodiesterases to G s -coupled β 2 -adrenergic receptors. Thus, β-arrestins can serve similar regulatory functions for disparate classes of 7TMRs through structurally dissimilar enzymes that degrade chemically distinct second messengers.

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol to form phosphatidic acid. In most cases, members of this large family of enzymes appear to bind and regulate proteins activated by either diacylglycerol or phosphatidic acid. Proteins that appear to be regulated, in part, by DGKs include protein kinase Cs, RasGRPs, and phosphatidylinositol kinases. By modulating the activity of these proteins, DGKs potentially affect a number of biological events including-but likely not limited to-cell growth, neuronal transmission, and cytoskeleton remodeling.

To study the physiological function of diacylglycerol (DAG) kinase (DGK ), which converts DAG to phosphatidic acid, we deleted this gene in mice. In contrast to previous studies showing that DGK isoforms decrease Ras activity, signaling downstream of Ras in embryonic fibroblasts was significantly reduced in cells lacking DGK . DGKs regulate Ras signaling by attenuating the function of the DAG-dependent Ras guanyl nucleotide-releasing proteins (RasGRPs). We tested whether DGK inhibited the four known RasGRPs and found that it inhibited only RasGRP3. In addition to activating Ras, RasGRP3 also activates Rap1, which in some cases can antagonize the function of Ras. We demonstrate that DGK bound to RasGRP3 and inhibited its activation of Rap1 by metabolizing DAG. This inhibition consequently affected Ras signaling. We tested the physiological consequence of deleting DGK by crossing wild-type or DGK -deficient mice with mice carrying a v-Ha-Ras transgene, and then we assessed tumor formation. We observed significantly fewer tumors in DGK -deficient mice. Because Rap1 can antagonize the function of Ras, our data are consistent with a model in which DGK regulates RasGRP3 with a predominant effect on Rap1 activity. Additionally, we found that DGK , which is structurally similar to DGK , inhibited RasGRPs 1, 3, and 4 and predominantly affected Ras signaling. Thus, type IV DGKs regulate RasGRPs, but the downstream effects differ depending on the DGK. D iacylglycerol (DAG) is a potent activator of several signaling proteins, many of which, when abnormally active, can contribute to the initiation or promotion of cancer (1, 2). Several enzymes can metabolize signaling DAG, but the major route is thought to be by its phosphorylation, a reaction that produces phosphatidic acid and is catalyzed by the DAG kinases (DGKs) (reviewed in ref. 2). DAG exerts its effects by activating classical and novel protein kinase C isoforms, as well as other proteins including the Ras guanyl nucleotide-releasing proteins (RasGRPs) (reviewed in refs. 3-5). Although the transforming effects of DAG have been attributed to activation of PKCs, identification of the RasGRP family suggested that DAG could also transform cells by directly activating RasGRPs, which in turn could lead to excess Ras signaling. The four known RasGRP proteins are RasGRP1 [calcium DAG guanine exchange factor II (CalDAG-GEFII)], RasGRP2 (CalDAG-GEFI), RasGRP3 (CalDAG-GEFIII), and RasGRP4 (reviewed in ref. 5). Each RasGRP activates either Ras or Rap1, except RasGRP3, which is unique because it facilitates exchange for both .Activating mutations in Ras are found in a number of tumors (reviewed in ref. 11). Using a mouse model, Chin et al. (12) showed that Ras expression was an absolute requirement for melanoma tumor maintenance, and other studies have demonstrated a role for Ras in metastasis (reviewed in ref. 13). Together, these data clearly show that abnormally active Ras contributes to the maintenance and progression of many types of cancer.Rap1-dependent signaling is not a...