Anders Heding

Development of a BRET2 Screening Assay Using β-Arrestin 2 Mutants

This study has focused on enhancing the signal generated from the interaction between a G-protein-coupled receptor (GPCR) and β-arrestin 2 (β-arr2), measured by the bioluminescence resonance energy transfer (BRET2) technology. Both class A (β2-adrenergic receptor [β2-AR]) and class B (neurokinin-type 1 receptor [NK1-R]) GPCRs, classified based on their internalization characteristics, have been analyzed. It was evaluated whether the BRET2 signal can be enhanced by using (1) β-arr2 phosphorylation-independent mutant (β-arr2 R169E) and (2) β-arr2 mutants deficient in their ability to interact with the components of the clathrin-coated vesicles (β-arr2 R393E, R395E and β-arr2 373 stop). For the class B receptor, there was no major difference in the agonist-promoted BRET2 signal when comparing results obtained with wild-type (wt) and mutant β-arr2. However, with the class A receptor, a more than 2-fold increase in the BRET2 signal was observed with β-arr2 mutants lacking the AP-2 or both AP-2 and clathrin binding sites. This set of data suggests that the inability of these β-arr2 mutants to interact with the components of the clathrin-coated vesicle probably prevents their rapid dissociation from the receptor, thus yielding an increased and more stable BRET2 signal. The β-arr2 R393E, R395E mutant also enhanced the signal window with other members of the GPCR family (neuropeptide Y type 2 receptor [NPY2-R] and TG1019 receptor) and was successfully applied in full-plate BRET2-based agonist and antagonist screening assays.

Negative cooperativity in H2 relaxin binding to a dimeric relaxin family peptide receptor 1

H2 relaxin, a member of the insulin superfamily, binds to the G-protein-coupled receptor RXFP1 (relaxin family peptide 1), a receptor that belongs to the leucine-rich repeat (LRR)-containing subgroup (LGRs) of class A GPCRs. We recently demonstrated negative cooperativity in INSL3 binding to RXFP2 and showed that this subgroup of GPCRs functions as constitutive dimers. In this work, we investigated whether the binding of H2 relaxin to RXFP1 also shows negative cooperativity, and whether this receptor functions as a dimer using BRET(2). Both binding and dissociation were temperature dependent, and the pH optimum for binding was pH 7.0. Our results showed that RXFP1 is a constitutive dimer with negative cooperativity in ligand binding, that dimerization occurs through the 7TM domain, and that the ectodomain has a stabilizing effect on this interaction. Dimerization and negative cooperativity appear to be general properties of LGRs involved in reproduction as well as other GPCRs.

Internalization kinetics of the gonadotropin-releasing hormone (GnRH) receptor.

This study quantified the agonist-induced endocytotic and recycling events of the mammalian gonadotropin releasing hormone receptor (GnRH-R) and investigated the role of the intracellular carboxyl (C)-terminal tail in regulating agonist-induced receptor internalization kinetics. The rate of internalization for the rat GnRH-R was found to be exceptionally low when compared with G-protein coupled receptors (GPCRs) which possess a cytoplasmic C-terminal tail (thyrotropin-releasing hormone receptor (TRH-R), catfish GnRH-R (cfGnRH-R) and GnRH/TRH-R chimeric receptor). These data provide evidence that the presence of a functional intracellular cytoplasmic C-terminal tail is essential for rapid internalization of the studied GPCRs.

Opsin Oligomerization in a Heterologous Cell System

Using bioluminescence resonance energy transfer (BRET) we studied opsin oligomerization in heterologous expression systems and quantitatively assessed its oligomerization state. BRET2 saturation and competition experiments were performed with live COS-7 cells expressing Rluc-and GFP2-tagged receptor constructs. BRET2 saturation curves obtained were hyperbolic, and the calculated oligomerization state (N = 1 for dimers) suggested that opsin (N = 1.34 ± 0.25) forms higher oligomers. Very high BRET2 values obtained for the opsin homo-dimer pair indicated a large energy transfer efficiency (E) and for cases where E ≫ 0.1 a modified saturation curve was proposed. The existence of homo-dimer complexes was additionally supported by competition assay results and was also observed in HEK-293 cells. Furthermore, evidence was provided for homo-and hetero-dimerization of family A (β2-adrenergic) and B (gastric inhibitory polypeptide, GIP) receptors. In summary, these experiments demonstrate homo-and hetero-dimerization for opsin, β 2-adrenergic, and GIP receptors.

Bioluminescence Resonance Energy Transfer as a Screening Assay: Focus on Partial and Inverse Agonism

The reported data for compound screening with the bioluminescence resonance energy transfer (BRET2) assay is very limited, and several questions remain unaddressed, such as the behavior of agonists. Eleven β2 adrenergic receptor (β2-AR) agonists were tested for full or partial agonism in an improved version of the receptor/β-arrestin2 BRET2 assay and in 2 cyclic adenosine monophosphate (cAMP) assays (column cAMP assay and ALPHAscreen™ cAMP assay). Tested in the highly sensitive ALPHAscreen™ cAMP assay, all selected agonists behaved as full agonists, using isoproterenol as a reference compound. In the less sensitive column cAMP assay, ephedrine and dopamine had a clear partial response. For the BRET2 assay, a highly graded picture was obtained. Moreover, β2-AR antagonists were tested for inverse agonism. Pronounced inverse agonism was detected in the ALPHAscreen™ cAMP assay. Only marginal inverse agonistic responses were seen for alprenolol and pindolol in the column cAMP assay, and no inverse agonism was seen in the BRET2 assay. For the β2-AR, the BRET2 assay may be superior for secondary screening of agonists where a separation of full and partial agonists is needed and the ALPHAscreen™ cAMP assay may be preferred for primary screening of agonists where all receptor activating compounds are desired.

Characterization of G-Protein Coupled Receptor Kinase Interaction with the Neurokinin-1 Receptor Using Bioluminescence Resonance Energy Transfer

To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer2 (BRET2) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive mutants. We observed agonist induced interaction of both GRK5 and GRK2 with the activated NK-1 receptor. In saturation experiments, we observed GRK5 to interact with the activated receptor in a monophasic manner while GRK2 interacted in a biphasic manner with the low affinity phase corresponding to receptor affinity for GRK5. Agonist induced GRK5 interaction with the receptor was dependent on intact kinase-activity, whereas the high affinity phase of GRK2 interaction was independent of kinase activity. We were surprised to find that the BRET2 saturation experiments indicated that before receptor activation, the full-length NK-1 receptor, but not a functional C-terminal tail-truncated receptor, is preassociated with GRK5 in a relatively low-affinity state. We demonstrate that GRK5 can compete for agonist induced GRK2 interaction with the NK-1 receptor, whereas GRK2 does not compete for receptor interaction with GRK5. We suggest that GRK5 is preassociated with the NK-1 receptor and that GRK5, rather than GRK2, is a key player in competitive regulation of GRK subtype specific interaction with the NK-1 receptor.

Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization, efficient signaling, and interaction with β-arrestin 2

This study was focused on the relationship between the plasma-membrane localization of neurokinin-1 receptor (NK1-R) and its endocytic and signaling properties. First, we employed electron paramagnetic resonance (EPR) to study the domain structure of HEK-293 cells and NK1-R microlocalization. EPR spectra and the GHOST condensation routine demonstrated that NK1-R was distributed in a well-ordered domain of HEK-293 cells possibly representing lipid raft/caveolae microdomains, whereas the impairment of caveolae changed the NK1-R plasma-membrane distribution. Internalization and second messenger assays combined with bioluminescence resonance energy transfer were employed subsequently to evaluate the functional importance of the NK1-R microlocalization in lipid raft/caveolae microdomains. The internalization pattern was delineated through the use of dominant-negative mutants (DNM) of caveolin-1 S80E (Cav1 S80E), dynamin-1 K44A (Dyn K44A), and β-arrestin (β-arr 319–418) and by means of cell lines that expressed various endogenous levels of β-arrestins. NK1-R displayed rapid internalization that was substantially reduced by DNMs of dynamin-1 and β-arrestin and even more profoundly in cells lacking both β-arrestin1 and β-arrestin2. These internalization data were highly suggestive of the predominant use of the clathrin-mediated pathway by NK1-R, even though NK1-R tended to reside constitutively in lipid raft/caveolae microdomains. Evidence was also obtained that the proper clustering of the receptor in these microdomains was important for effective agonist-induced NK1-R signaling and for its interaction with β-arrestin2.

Novel method for the study of receptor Ca2+ signalling exemplified by the NK1 receptor.

ABSTRACT We have used a novel technology (NovoStar from BMG Labtechnologies) for the study of the Ca2+ signalling of the human tackykinin NK1 (hNK-1 receptor). The NovoStar is a microplate reader based on fluorescence and luminescence. The instrument implements a robotic pipettor arm and two microplate carriers, typically one for samples and one for cells. The robotic pipettor arm can transfer sample (agonist or antagonist) from the sample plate or other liquid containers to the cell plate, facilitating the study of Ca2+ signalling to such a degree that the instrument can be used for Medium Throughput Screening (MTS). Using the NovoStar we have found the molecular pharmacology of the NK1 receptor to be comparable to that observed in classical signal transduction assays. Thus, we have observed an EC50 value of 3 nM for substance P induced Ca2+ response. This value corresponds well with previously published values for substance P induced IP and cAMP turnover. Using the NovoStar technology we have studied the pharmacological profile of the well known non-peptide NK1 receptor antagonists CP96,345 and SR140,333 in respect of inhibition of the Ca2+ response induced by substance P. Interestingly, the antagonistic potency of the antagonists depended greatly on the experimental design, e.g., a dependency of timing in the addition of antagonists vs. agonist was noted. Also, metal-ion site engineered NK1 receptors were tested for the ability of metal-ions to inhibit signalling. It is concluded that the NovoStar is a reliable tool for the study of receptor Ca2+ signalling, both as a research tool and as a MTS system.

Dimerization and Negative Cooperativity in the Relaxin Family Peptide Receptors

Peptides of the relaxin family bind to the relaxin family peptide receptors or RXFPs, members of the G‐protein‐coupled receptor (GPCR) superfamily. For many years, ligand binding to GPCRs was thought to take place as monomeric complexes, ignoring early evidence of negative cooperativity. However, recent research has shown that most GPCRs form constitutive dimers or larger oligomers. The connection between dimerization and negative cooperativity has now been shown for several GPCRs, including the thyroid‐stimulating hormone, luteinizing hormone, and follicle‐stimulating hormone receptors, which like RXFP1 and ‐2 belong to the leucine‐rich repeat‐containing subgroup of class A GPCRs. We recently demonstrated homodimerization and negative cooperativity for RXFP1 and RXFP2 as well as their heterodimerization. Another study showed that RXFP1 has to homodimerize in order to be transported from the endoplasmic reticulum to the cell membrane.