Alteration in [Ca(2+)](i) (the intracellular concentration of Ca(2+)) is a key regulator of many cellular processes. To allow precise regulation of [Ca(2+)](i) and a diversity of signalling by this ion, cells possess many mechanisms by which they are able to control [Ca(2+)](i) both globally and at the subcellular level. Among these are many members of the superfamily of GPCRs (G-protein-coupled receptors), which are characterized by the presence of seven transmembrane domains. Typically, those receptors able to activate PLC (phospholipase C) enzymes cause release of Ca(2+) from intracellular stores and influence Ca(2+) entry across the plasma membrane. It has been well documented that Ca(2+) signalling by one type of GPCR can be influenced by stimulation of a different type of GPCR. Indeed, many studies have demonstrated heterologous desensitization between two different PLC-coupled GPCRs. This is not surprising, given our current understanding of negative-feedback regulation and the likely shared components of the signalling pathway. However, there are also many documented examples of interactions between GPCRs, often coupling preferentially to different signalling pathways, which result in a potentiation of Ca(2+) signalling. Such interactions have important implications for both the control of cell function and the interpretation of in vitro cell-based assays. However, there is currently no single mechanism that adequately accounts for all examples of this type of cross-talk. Indeed, many studies either have not addressed this issue or have been unable to determine the mechanism(s) involved. This review seeks to explore a range of possible mechanisms to convey their potential diversity and to provide a basis for further experimental investigation.

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Both homo-and heterodimeric interactions between the CXCR1 and CXCR2 chemokine receptors were observed following co-expression of forms of these receptors in HEK293 cells using assays, including co-immunoprecipitation, single cell imaging of fluorescence resonance energy transfer, cell surface time-resolved fluorescence resonance energy transfer, and bioluminescence resonance energy transfer. These interactions were constitutive and unaffected by the presence of the agonist interleukin 8 and selective as no significant interactions were noted between either the CXCR1 or CXCR2 receptor and the ␣ 1A -adrenoreceptor. Saturation bioluminescence resonance energy transfer indicated that heteromeric interactions between CXCR1 and CXCR2 were of similar affinity as the corresponding homomeric interactions. A novel endoplasmic reticulum trapping strategy demonstrated that these interactions were initiated during protein synthesis and maturation and prior to cell surface delivery. These studies indicated that CXCR1-CXCR2 heterodimers are as likely to form in cells co-expressing these two chemokine receptors as the corresponding homodimers and stand in contrast to previous studies indicating an inability of the CXCR1 receptor to homodimerize or to interact with the CXCR2 receptor (Trettel, F., Di Bartolomeo, S., Lauro, C., Catalano, M., Ciotti, M. T., and Limatola, C. (2003) J. Biol. Chem. 278, 40980 -40988).In recent times the concept that G protein-coupled receptors (GPCRs) 1 can exist as dimers and/or higher order oligomers has become increasingly accepted (1-5). Techniques ranging from the co-immunoprecipitation of co-expressed but differentially epitope-tagged forms of a single receptor species (6, 7) to the use of resonance energy transfer-based methods (8 -12) have provided support for the presence of such dimers/oligomers for many GPCRs in transfected cell systems, and the application of atomic force microscopy has shown the presence of dimers and arrays of dimers of rhodopsin in murine rod outer segment discs (13,14). In many cases, GPCR quaternary structure seems to be defined early in the processes of receptor synthesis and maturation with the GPCR being transported to the cell surface as a preformed dimer/oligomer (15-17), the structure of which is unaffected by the presence of agonist ligands. The potential quaternary structure of a range of chemokine receptors has also been explored by using similar approaches (18 -23). Although a substantial number of chemokine receptors have been shown to possess such quaternary structure, a number of features of certain chemokine receptors are either controversial or seem not to follow the general model outlined above. For example, dimerization/oligomerization of a number of chemokine receptors appears to be promoted by the binding of chemokine ligands (18,19). Equally, it appears that mutation of certain chemokine receptors to prevent dimerization does not restrict membrane delivery (24). Among the chemokine receptors (25), the closely related CXCR1 and CXCR2 receptors sha...

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9-39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9-39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

Glucagon-like peptide-1 (GLP-1) mediates antidiabetogenic effects through the GLP-1 receptor (GLP-1R), which is targeted for the treatment of type 2 diabetes. Small-molecule GLP-1R agonists have been sought due to difficulties with peptide therapeutics. Recently, 6,7-dichloro-2-methylsulfonyl-3-N-tertbutylaminoquinoxaline (compound 2) has been described as a GLP-1R allosteric modulator and agonist. Using human embryonic kidney-293 cells expressing human GLP-1Rs, we extended this work to consider the impact of compound 2 on G protein activation, Ca 2ϩ signaling and receptor internalization and particularly to compare compound 2 and GLP-1 across a range of functional assays in intact cells. GLP-1 and compound 2 activated G␣ s in cell membranes and increased cellular cAMP in intact cells, with compound 2 being a partial and almost full agonist, respectively. GLP-1 increased intracellular [Ca 2ϩ ] by release from intracellular stores, which was mimicked by compound 2, with slower kinetics. In either intact cells or membranes, the orthosteric antagonist exendin-(9-39), inhibited GLP-1 cAMP generation but increased the efficacy of compound 2. GLP-1 internalized enhanced green fluorescent protein-tagged GLP-1Rs, but the speed and magnitude evoked by compound 2 were less. Exendin-(9-39) inhibited internalization by GLP-1 and also surprisingly that by compound 2. Compound 2 displays GLP-1R agonism consistent with action at an allosteric site, although an orthosteric antagonist increased its efficacy on cAMP and blocked compound 2-mediated receptor internalization. Full assessment of the properties of compound 2 was potentially hampered by damaging effects that were particularly manifest in either longer term assays with intact cells or in acute assays with membranes.

1 We have examined the phospholipase C responses in bovine aortic endothelial cells to purines (ATP, ADP and analogues) and the pyrimidine, uridine triphosphate (UTP).2 The cells responded to purines in a manner consistent with the presence of P2y purinoceptors; both 2-methylthioadenosine 5'-triphosphate (2MeSATP) and adenosine 5'-0-(2-thiodiphosphate) (ADPPS) were potent agonists (ECm0 0.41 gtM and 0.85 llM respectively) while P, y-methylene ATP at 300 $1M was not.3 The cells also responded to UTP. The maximal response to UTP was less than that for either 2MeSATP and ADPI3S while adenosine 5'-0-(3-thiotriphosphate) (ATPyS) gave the largest maximal response. 4 The concentration-effect curve to UTP was additive in the presence of either 2MeSATP or ADPPS.However, the concentration-effect curves to ATPyS reached the same maximum in the presence or absence of UTP. 5 Suramin, at concentrations between 10 lM and 100 gsM was a competitive antagonist for the response to ADPPS and 2MeSATP but not the response to UTP. 6 The results show that there are two separate, co-existing, receptor populations: P2y-purinoceptors (responding to purines) and nucleotide receptors (responding to both purines and pyrimidines). We conclude that purines such as ATP/ADP may regulate aortic endothelial cells by interacting with two phospholipase C-linked receptors.

Background and purpose:The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B of the G protein-coupled receptor superfamily and is a target for treatment of type 2 diabetes. Family B G protein-coupled receptors contain a putative N-terminal signal peptide, but its role in receptor synthesis and trafficking are unclear. Further, the signal peptide is not cleaved in at least one family member. Experimental approach: We examined receptor glycosylation and the role of the signal peptide in GLP-1R synthesis and trafficking using constructs containing epitope tags at the N-and/or C-terminus and in which the signal peptide sequence was either present or absent. Key results: The signal peptide was absolutely required for GLP-1R synthesis but could be substituted to some extent by increasing positive charge in the N-terminal region of the receptor flanking the signal peptide. The signal peptide is cleaved during synthesis and processing of the receptor. An enhanced GFP-epitope tag at the N-terminus of the receptor permitted synthesis of the receptor but blocked signal peptide cleavage and prevented trafficking to the plasma membrane. Cleavage site mutation allowed synthesis of a full-length receptor, blocked signal peptide cleavage and caused retention within the endoplasmic reticulum. Conclusions and implications:Signal peptide cleavage was not essential for receptor synthesis but was obligatory for processing and trafficking of receptors to the plasma membrane. Further, the GLP-1R is subject to N-linked glycosylation and only the mature, fully glycosylated form of the receptor is present in the plasma membrane. Inhibition of glycosylation prevents processing and cell surface expression of the GLP-1R. Doyle and Egan, 2007). Enhancing GLP-1R-mediated effects through either inhibition of the rapid breakdown of GLP-1 by dipeptidyl peptidase-IV or by direct activation of the GLP-1R is now a validated therapeutic option for the treatment of type 2 diabetes. The GLP-1R belongs to the relatively small Family B of the G-protein-coupled receptor (GPCR) superfamily. Despite being integral membrane proteins with seven transmembrane a-helices, a large proportion of GPCRs (~85%) display no evidence of a signal peptide sequence (Köchl et al., 2002). This is a sequence of approximately 20 amino acids containing a stretch of hydrophobic residues, usually in the N-terminus of the protein, which is often critical for the synthesis and processing of both secreted and integral membrane proteins. However, these signal peptide sequences are removed by signal peptidases within the endoplasmic reticulum. In GPCRs without such a sequence, the first transmembrane domain can function as a signal anchor sequence, promoting post-translational movement of the N-terminus through the translocon into the lumen of the endoplasmic reticulum during which time the nascent peptide may be subject to N-linked glycosylation. Subsequently, signal anchor sequences within the receptor transmembrane domains are thought to regulate further synthesis and...

H]-InsPx accumulation was inhibited by increasing concentrations of pertussis toxin (0.01 ± 100 ng ml 71 ), indicating the involvement of pertussis toxin-sensitive G-proteins. 6 These ®ndings show that the human recombinant sst 5 receptor, when stably expressed in CHO-K1 cells, is able to mediate activation of phosphoinositide metabolism in a pertussis toxin-sensitive manner. In this system L-362,855 behaved as a partial agonist while BIM-23056 was a speci®c antagonist. These agents should provide useful tools for functionally characterizing endogenous SRIF receptors.