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There is increasing complexity in the organization of seven transmembrane domain (7TM) receptor signalling pathways, and in the ability of their ligands to modulate and direct this signalling. Underlying these events is a network of protein interactions between the 7TM receptors themselves and associated effectors, such as G proteins and beta-arrestins. Bimolecular fluorescence complementation, or BiFC, is a technique capable of detecting these protein-protein events essential for 7TM receptor function. Fluorescent proteins, such as those from Aequorea victoria, are split into two non-fluorescent halves, which then tag the proteins under study. On association, these fragments refold and regenerate a mature fluorescent protein, producing a BiFC signal indicative of complex formation. Here, we review the experimental criteria for successful application of BiFC, considered in the context of 7TM receptor signalling events such as receptor dimerization, G protein and beta-arrestin signalling. The advantages and limitations of BiFC imaging are compared with alternative resonance energy transfer techniques. We show that the essential simplicity of the fluorescent BiFC measurement allows high-content and advanced imaging applications, and that it can probe more complex multi-protein interactions alone or in combination with resonance energy transfer. These capabilities suggest that BiFC techniques will become ever more useful in the analysis of ligand and 7TM receptor pharmacology at the molecular level of protein-protein interactions.

The ghrelin receptor (GhrelinR) and its related orphan GPR39 each display constitutive signaling, but only GhrelinRs undergo basal internalization. Here we investigate these differences by considering the roles of the C tail receptor domains for constitutive internalization and activity. Furthermore the interaction between phosphorylated receptors and β-arrestin adaptor proteins has been examined. Replacement of the FLAG-tagged GhrelinR C tail with the equivalent GPR39 domain (GhR-39 chimera) preserved Gq signaling. However in contrast to the GhrelinR, GhR-39 receptors exhibited no basal and substantially decreased agonist-induced internalization in transiently transfected HEK293 cells. Internalized GhrelinR and GhR-39 were predominantly localized to recycling compartments, identified with transferrin and the monomeric G proteins Rab5 and Rab11. Both the inverse agonist [d-Arg1, d-Phe5, d-Trp7,9, Leu11] substance P and a naturally occurring mutant GhrelinR (A204E) with eliminated constitutive activity inhibited basal GhrelinR internalization. Surprisingly, we found that noninternalizing GPR39 was highly phosphorylated and that basal and agonist-induced phosphorylation of the GhR-39 chimera was elevated compared with GhrelinRs. Moreover, basal GhrelinR endocytosis occurred without significant phosphorylation, and it was not prevented by cotransfection of a dominant-negative β-arrestin1(319–418) fragment or by expression in β-arrestin1/2 double-knockout mouse embryonic fibroblasts. In contrast, agonist-stimulated GhrelinRs recruited the clathrin adaptor green fluorescent protein-tagged β-arrestin2 to endosomes, coincident with increased receptor phosphorylation. Thus, GhrelinR internalization to recycling compartments depends on C-terminal motifs and constitutive activity, but the high levels of GPR39 phosphorylation, and of the GhR-39 chimera, are not sufficient to drive endocytosis. In addition, basal GhrelinR internalization occurs independently of β-arrestins.

The use of the click reaction for the introduction of conjugate groups, such as affinity or fluorescent labels, to a peptide for the study of peptide biochemistry and pharmacology is widespread. However, the nature and location of substituted 1,2,3-triazoles in peptide sequences may markedly affect conformation or binding as compared with native sequences. We have examined the preparation and application of propargyloxyproline (Pop) residues as a precursor to such peptide conjugates. Pop residues are available in a range of regio- and stereoisomers from hydroxyproline precursors and are readily prepared in Fmoc-protected form. They can be incorporated routinely in peptide synthesis and broadly retain the conformational properties of the parent proline containing peptides. This is exemplified by the preparation of biotin- and fluorophore-labelled peptides derived from linear and cyclic peptides.

The antisecretory effects of several Y agonists, including pancreatic polypeptide (PP), indicate the presence of Y(1), Y(2), and Y(4) receptors in mouse and human (h) colon mucosae. Here, we used preparations from human and from wild-type (WT), Y(4), and Y(1) receptor knockout ((-/-)) mice, alongside Y(4) receptor-transfected cells to define the relative functional contribution of the Y(4) receptor. First, rat (r) PP antisecretory responses were lost in murine Y(4)(-/-) preparations, but hPP and Pro(34) peptide YY (PYY) costimulated Y(4) and Y(1) receptors in WT mucosa. The Y(1) antagonist/Y(4) agonist GR231118 [(Ile,Glu,Pro,Dpr,Tyr,Arg,Leu,Arg,Try-NH(2))-2-cyclic(2,4'),(2',4)-diamide] elicited small Y(4)-mediated antisecretory responses in human tissues pretreated with the Y(1) antagonist, BIBO3304 [(R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N(2)-(diphenylacetyl)-argininamide trifluoroacetate)], and attenuated Y(4)-mediated hPP responses in mouse and human mucosa. GR231118 and rPP were also antisecretory in hY(4)-transfected epithelial monolayers but were partial agonists compared with hPP at this receptor. In Y(4)-transfected human embryonic kidney (HEK) 293 cells, Y(4) ligands displaced [(125)I]hPP binding with orders of affinity (pK(i)) at human (hPP = rPP > GR231118 > Pro(34)PYY = PYY) and mouse (rPP = hPP > GR231118 > Pro(34)PYY > PYY) Y(4) receptors. GR231118- and rPP-stimulated guanosine 5'-3-O-(thio)triphosphate binding through hY(4) receptors with significantly lower efficacy than hPP. GR231118 marginally increased basal but abolished further PP-induced hY(4) internalization to recycling (transferrin-labeled) pathways in HEK293 cells. Taken together, these findings show that Y(4) receptors play a definitive role in attenuating colonic anion transport and may be useful targets for novel antidiarrheal agents due to their limited peripheral expression.

The effects of a number of agonists which inhibit intestinal chloride secretion were investigated in Colony-1 (Col-1) cells, a subpopulation derived from the HCA-7 human adenocarcinoma cell line. Neither peptide YY (PYY) or somatostatin 14-28 (SRIF) reduced short-circuit current (SCC) in Col-1 epithelial layers stimulated with vasoactive intestinal polypeptide (VIP), suggesting that their respective receptors are either absent in this cell line, or are not functionally coupled. A second member of the neuropeptide Y family, pancreatic polypeptide (PP), decreased VIP-elevated SCC with an EC50 of 25.6 nM. Maximal PP responses were unaffected by prior addition of PYY, indicating that Col-1 cells may express a PP specific, Y4-like receptor. The alpha 2-adrenoceptor agonist clonidine also attenuated VIP-stimulated SCC (EC50342 nM) through the alpha 2A receptor subtype, since clonidine responses were inhibited by yohimbine and rauwolscine but not altered by previous addition of prazosin. Col-1 cells responded to both apical and basolateral addition of VIP or clonidine; to an extent, this lack of sidedness reflects the ability of drugs to permeate through the Col-1 epithelial layers. Both PP and clonidine also inhibited SCC in unstimulated Col-1 cells or those pretreated with 3-isobutyl-1-methylaxanthine (IBMX) or a submaximal concentration of forskolin, agents which both directly elevate intracellular cAMP. After a maximal concentration of forskolin (10 microM), which increased SCC to a significantly greater extent than either VIP or IBMX, the effects of both agonists were negligible. The absence of PP and clonidine responses under these conditions may have implications for the mechanisms by which these agonists inhibit, chloride secretion in Col-1 epithelia. In addition carbachol reduced SCC stimulated by 10 microM forskolin, in contrast to control carbachol responses which consisted of a rapid decrease followed by a transient elevation in SCC; this observation suggests that Col-1 cells may also be a useful model for studying the interactions between Ca(2+)- and cAMP-dependent mechanisms involved in epithelial ion transport.

Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis are powerful ways to study mobility and stoichiometry of G protein coupled receptor complexes, within microdomains of single living cells. However, relating these properties to molecular mechanisms can be challenging. We investigated the influence of β-arrestin adaptors and endocytosis mechanisms on plasma membrane diffusion and particle brightness of GFP-tagged neuropeptide Y (NPY) receptors. A novel GFP-based bimolecular fluorescence complementation (BiFC) system also identified Y1 receptor-β-arrestin complexes. Diffusion co-efficients (D) for Y1 and Y2-GFP receptors in HEK293 cell plasma membranes were 2.22 and 2.15 × 10− 9 cm2 s− 1 respectively. At a concentration which promoted only Y1 receptor endocytosis, NPY treatment reduced Y1-GFP motility (D 1.48 × 10− 9 cm2 s− 1), but did not alter diffusion characteristics of the Y2-GFP receptor. Agonist induced changes in Y1 receptor motility were inhibited by mutations (6A) which prevented β-arrestin recruitment and internalisation; conversely they became apparent in a Y2 receptor mutant with increased β-arrestin affinity. NPY treatment also increased Y1 receptor-GFP particle brightness, changes which indicated receptor clustering, and which were abolished by the 6A mutation. The importance of β-arrestin recruitment for these effects was illustrated by reduced lateral mobility (D 1.20–1.33 × 10− 9 cm2 s− 1) of Y1 receptor-β-arrestin BiFC complexes. Thus NPY-induced changes in Y receptor motility and brightness reflect early events surrounding arrestin dependent endocytosis at the plasma membrane, results supported by a novel combined BiFC/FCS approach to detect the underlying receptor-β-arrestin signalling complex., Graphical abstract Highlights ► First FCS / PCH analysis of the plasma membrane diffusion characteristics of NPY receptors. ► Demonstration that NPY changes in receptor diffusion and oligomeric state depend on early events in endocytosis. ► Design of a new GFP BiFC system for use in combination with FCS techniques. ► Isolation of the mobility and molecular brightness of defined receptor-arrestin complexes undergoing endocytosis.