In the present work, we studied LPA2-mediated signaling using human colon cancer cell lines, which predominantly express LPA2. LPA2 activated Akt and Erk1/2 in response to LPA. LPA mediated Akt activation was inhibited by pertussis toxin (PTX), whereas Erk1/2 activation was completely inhibited by a blocker of phospholipase C␤, U-73122. LPA also induced interleukin-8 (IL-8) synthesis in the colon cancer cells by primarily activating LPA2 receptor. We also found that LPA2 interacts with Na ϩ /H ϩ exchanger regulatory factor 2 (NHERF2). Activation of Akt and Erk1/2 was significantly attenuated by silencing of NHERF2 expression by RNA interference, suggesting a pivotal role of NHERF2 in LPA2-mediated signaling. We found that expression of LPA2 was elevated, whereas expression of LPA1 downregulated in several types of cancers, including ovarian and colon cancer. We conclude that LPA2 is the major LPA receptor in colon cancer cells and cellular signals by LPA2 are largely mediated through its ability to interact with NHERF2.

Many G protein-coupled receptors possess carboxyl-terminal motifs ideal for interaction with PDZ scaffold proteins, which can control receptor trafficking and signaling in a cell-specific manner. To gain a panoramic view of ␤ 1 -adrenergic receptor (␤ 1 AR) interactions with PDZ scaffolds, the ␤ 1 AR carboxyl terminus was screened against a newly developed proteomic array of PDZ domains. These screens confirmed ␤ 1 AR associations with several previously identified PDZ partners, such as PSD-95, MAGI-2, GIPC, and CAL. Moreover, two novel ␤ 1 AR-interacting proteins, SAP97 and MAGI-3, were also identified. The ␤ 1 AR carboxyl terminus was found to bind specifically to the first PDZ domain of MAGI-3, with the last four amino acids (E-S-K-V) of ␤ 1 AR being the key determinants of the interaction. Full-length ␤ 1 AR robustly associated with full-length MAGI-3 in cells, and this association was abolished by mutation of the ␤ 1 AR terminal valine residue to alanine (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. MAGI-3 co-expression with ␤ 1 AR profoundly impaired ␤ 1 AR-mediated ERK1/2 activation but had no apparent effect on ␤ 1 AR-mediated cyclic AMP generation or agonist-promoted ␤ 1 AR internalization. These findings revealed that the interaction of MAGI-3 with ␤ 1 AR can selectively regulate specific aspects of receptor signaling. Moreover, the screens of the PDZ domain proteomic array provide a comprehensive view of ␤ 1 AR interactions with PDZ scaffolds, thereby shedding light on the molecular mechanisms by which ␤ 1 AR signaling and trafficking can be regulated in a cell-specific manner.Cellular responses to a given hormone or neurotransmitter can vary markedly between different types of cells. In many cases, such cellular differences are because of differential expression of receptor subtypes. However, even cells expressing exactly the same receptor subtypes can exhibit distinct responses to a given ligand, because receptors often behave quite differently in distinct cellular environments. The trafficking and signaling properties of G protein-coupled receptors (GPCRs), 3 which comprise the largest family of cell surface receptors, are known to be especially influenced by cellular context. The activity of a GPCR depends not only on the complement of downstream effectors expressed in a given cell but also on the set of expressed G proteins, kinases, and scaffold proteins that directly interact with the receptor to shape its signaling capability.PDZ scaffolds comprise a key class of GPCR-interacting proteins that can strongly influence receptor trafficking and signaling. The term PDZ is derived from the names of the first three proteins in which these domains were first identified: the post-synaptic density protein PSD-95, the Drosophila protein Discs-large, and the tight junction protein ZO-1. PDZ domains are ϳ90 amino acids in length and bind to specific carboxyl-terminal motifs on their target proteins. There are three general classes of PDZ domain...

P2Y1 purinergic receptors (P2Y1Rs) mediate rises in intracellular Ca 2؉ in response to ATP, but the duration and characteristics of this Ca 2؉ response are known to vary markedly in distinct cell types. We screened the P2Y1R carboxyl terminus against a recently created proteomic array of PDZ (PSD-95͞Drosophila Discs large͞ZO-1 homology) domains and identified a previously unrecognized, specific interaction with the second PDZ domain of the scaffold NHERF-2 (Na ؉ ͞H ؉ exchanger regulatory factor type 2). Furthermore, we found that P2Y1R and NHERF-2 associate in cells, allowing NHERF-2-mediated tethering of P2Y 1R to key downstream effectors such as phospholipase C␤. Finally, we found that coexpression of P2Y1R with NHERF-2 in glial cells prolongs P2Y1R-mediated Ca 2؉ signaling, whereas disruption of the P2Y1R-NHERF-2 interaction by point mutations attenuates the duration of P2Y1R-mediated Ca 2؉ responses. These findings reveal that NHERF-2 is a key regulator of the cellular activity of P2Y 1R and may therefore determine cellspecific differences in P2Y1R-mediated signaling.G protein-coupled receptor ͉ purinergic ͉ ATP ͉ proteomic array A denine-based nucleotides such as ATP and ADP are prominent extracellular signaling molecules that mediate a wide variety of physiological actions in tissues throughout the body. Many of the physiological effects evoked by ATP and ADP are mediated by metabotropic P2Y receptors (P2YRs) (1), which are members of the G protein-coupled receptor (GPCR) superfamily. To date, seven distinct mammalian P2YR subtypes have been cloned: P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , and P2Y 13 . Most P2YRs are coupled to G␣ q proteins and thus to the activation of phospholipase C␤ (PLC␤) and generation of diacylglycerol and inositol-3,4,5-trisphosphate, ultimately leading to the activation of PKC and release of Ca 2ϩ from internal stores (1). Some P2YRs, including P2Y 1 , P2Y 2 , P2Y 12 , and P2Y 13 , are also known to couple to G␣ i and the inhibition of adenylyl cyclase (2-5). The purinergic P2YR type 1 (P2Y 1 R) subtype is abundantly expressed in a number of tissues, including the CNS (6, 7), where it plays a key role in the transmission of astrocytic Ca 2ϩ waves (8), activation of mitogenic responses in astrocytes to brain trauma (9), inhibition of neuronal N-type voltage-activated Ca 2ϩ channels (10), and embryonic brain development (11). P2Y 1 R also plays critical roles in the cardiovascular system, including the regulation of coronary vasodilation (12) and platelet aggregation (13).Signaling by P2Y 1 Rs is known to be heavily dependent on cellular context. For example, stimulation of P2Y 1 Rs in some cell types is known to strongly promote cell proliferation (14), whereas P2Y 1 R stimulation in other cell types is known to induce apoptosis (15). Furthermore, P2Y 1 Rs can exert cellular effects that are quite different from those exerted by other P2YRs expressed in the same cell type and that couple to similar G proteins. For example, in astrocytes, both P2Y 1 Rs and P2Y 2 Rs can cou...

The physiological actions of epinephrine and norepinephrine are mediated via the activation of the following three distinct classes of G protein-coupled receptors (GPCR) 1 : ␣ 1 -, ␣ 2 -, and ␤-adrenergic receptors. Each class of adrenergic receptor (AR) is comprised of three closely related subtypes as follows: ␣ 1A -, ␣ 1B -, and ␣ 1D AR, which couple primarily to G q to stimulate phospholipase activity; ␣ 2A -, ␣ 2B -, and ␣ 2C AR, which couple primarily to G i to inhibit adenylyl cyclase activity; and ␤ 1 -, ␤ 2 -, and ␤ 3 AR, which couple primarily to G s to stimulate adenylyl cyclase activity (1). The adrenergic receptor subtypes are differentially distributed across various tissues, and tissue responses to epinephrine and norepinephrine are believed to be dependent upon the relative ratios of the various adrenergic receptors they express.Because ␤-and ␣ 2 -adrenergic receptors couple to G proteins with opposing actions on adenylyl cyclase activity, the two receptors might be expected to purely antagonize each other's signaling when they are co-stimulated in the same cell. However, it has been shown that ␣ 2 AR co-stimulation can in some cases paradoxically sensitize ␤-adrenergic signaling in brain tissue (2-4). Moreover, the pharmacological properties of ␤ARs in brain tissue are known to be regulated by ␣ 2 ARs (5, 6), and reciprocally the pharmacological properties of ␣ 2 ARs in brain tissue are known to regulated by ␤ARs (7,8). These examples of cross-talk and mutual regulation between ␤-and ␣ 2 -adrenergic receptors have been well known for more than 20 years, but the underlying molecular mechanisms remain unclear.GPCRs have traditionally been thought to exist as monomers, but recent studies (9) have revealed that they can exist in the plasma membrane as both homodimers and heterodimers. At present, a key question in this field is: how widespread is the phenomenon of receptor heterodimerization? The most clearcut case of the importance of GPCR heterodimerization comes from the GABA B receptor, a pharmacologically defined entity that is now known to be comprised of two distinct GPCRs, GABA B R1 and GABA B R2 (10). Because GABA B R1 and GAB-A B R2 are not functional when expressed by themselves, they represent a clear example of the physiological importance of receptor heterodimerization. Although other heptahelical receptors may not absolutely require heterodimerization to be functional in the same way that the GABA B receptor does, heterodimerization of other receptors may underlie some phenomena that are major question marks in our present understanding of neurotransmitter and hormone receptors, such as unexplained forms of cross-talk between different receptor subtypes.We wondered if the previously reported cross-talk between ␤ARs and ␣ 2 ARs in brain tissue might be due in part to a physical association between these two receptor types. Many early studies (11-13) of GPCR dimerization focused on the ␤ 2 AR. We have found recently (14) that the ␤ 1 AR also exhibits robust homodimerization in cells. Fu...

G protein-coupled receptors such as the ␤1-adrenergic receptor (␤1AR) must be trafficked to the plasma membrane in order to bind with their extracellular ligands and regulate cellular physiology. By using glutathione S-transferase pull-down techniques, we found that the ␤1AR carboxyl terminus directly interacts with the cystic fibrosis transmembrane conductance regulator-associated ligand (CAL; also known as PIST, GOPC, and FIG), a protein known to be primarily localized to the Golgi apparatus. CAL contains two predicted coiled-coil domains and one PSD-95/Discs-large/ZO-1 homology (PDZ) domain. The ␤1AR carboxyl terminus (CT) binds to the PDZ domain of CAL, with the last few amino acids (ESKV) of the ␤1〈R-CT being the key determinants for the interaction. Mutation of the terminal valine residue resulted in markedly reduced association of the ␤1AR-CT with CAL. Numerous other mutations to the ESKV motif also impaired the ␤1AR-CT/CAL interaction, suggesting that this motif is close to optimal for association with the CAL PDZ domain. In cells, full-length ␤1AR robustly associates with CAL, and this interaction is abolished by mutation of the terminal valine to alanine of the receptor (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. Consistent with observations that CAL is a Golgi-associated protein, overexpression of CAL reduces surface expression of ␤1AR. Interaction with CAL promotes retention of ␤1AR within the cell, whereas PSD-95, another ␤1AR-associated PDZ domaincontaining protein, competitively blocks ␤1AR association with CAL and promotes receptor trafficking to the cell surface. These data reveal that CAL, a novel ␤1AR-binding partner, modulates ␤1AR intracellular trafficking, thereby revealing a new mechanism of regulation for ␤1AR anterograde trafficking through the endoplasmic reticulum-Golgi complex to the plasma membrane. ␤-Adrenergic receptors (␤ARs)1 are G protein-coupled receptors (GPCRs) that play critical roles in mediating physiological responses to the hormone epinephrine and the neurotransmitter norepinephrine. Noradrenergic stimulation of ␤1-adrenergic receptor (␤1AR) in the brain potently regulates memory formation and synaptic plasticity (1-6), and stimulation of ␤1AR in the heart profoundly regulates both the rate and force of cardiac contractions (7-9). Neither norepinephrine nor epinephrine can easily cross the plasma membrane; thus, ␤1AR must be localized at the cell surface in order to be activated by its ligands. The regulation of the trafficking and surface expression of ␤1AR and other GPCRs is therefore a topic of considerable physiological importance.Intracellular trafficking of GPCRs has been extensively studied, with most of this work focused on the events involved in receptor internalization and recycling. Within seconds or minutes of agonist stimulation, ␤ARs are phosphorylated by G protein-coupled receptor kinases and cAMP-dependent protein kinases (10, 11). Subsequently, in the cases of ␤1AR and ␤2AR, this phosphoryl...