Rasmus Jorgensen

Oxyntomodulin Differentially Affects Glucagon-Like Peptide-1 Receptor β-Arrestin Recruitment and Signaling through Gα

The glucagon-like peptide (GLP)-1 receptor is a promising target for the treatment of type 2 diabetes and obesity, and there is great interest in characterizing the pharmacology of the GLP-1 receptor and its ligands. In the present report, we have applied bioluminescence resonance energy transfer2 assays to measure agonist-induced recruitment of βarrestins and G-protein-coupled receptor kinase (GRK) 2 to the GLP-1 receptor in addition to traditional measurements of second messenger generation. The peptide hormone oxyntomodulin is described in the literature as a full agonist on the glucagon and GLP-1 receptors. Surprisingly, despite being full agonists in GLP-1 receptor-mediated cAMP accumulation, oxyntomodulin and glucagon were observed to be partial agonists in recruiting βarrestins and GRK2 to the GLP-1 receptor. We suggest that oxyntomodulin and glucagon are biased ligands on the GLP-1 receptor.

Metal Ion Site Engineering Indicates a Global Toggle Switch Model for Seven-transmembrane Receptor Activation

Much evidence indicates that, during activation of seven-transmembrane (7TM) receptors, the intracellular segments of the transmembrane helices (TMs) move apart with large amplitude, rigid body movements of especially TM-VI and TM-VII. In this study, AspIII:08 (Asp113), the anchor point for monoamine binding in TM-III, was used as the starting point to engineer activating metal ion sites between the extracellular segments of theβ2-adrenergic receptor. Cu(II) and Zn(II) alone and in complex with aromatic chelators acted as potent (EC50 decreased to 0.5 μm) and efficacious agonists in sites constructed between positions III:08 (Asp or His), VI:16 (preferentially Cys), and/or VII:06 (preferentially Cys). In molecular models built over the backbone conformation of the inactive rhodopsin structure, the heavy atoms that coordinate the metal ion were located too far away from each other to form high affinity metal ion sites in both the bidentate and potential tridentate settings. This indicates that the residues involved in the main ligand-binding pocket will have to move closer to each other during receptor activation. On the basis of the distance constraints from these activating metal ion sites, we propose a global toggle switch mechanism for 7TM receptor activation in which inward movement of the extracellular segments of especially TM-VI and, to some extent, TM-VII is coupled to the well established outward movement of the intracellular segments of these helices. We suggest that the pivots for these vertical seesaw movements are the highly conserved proline bends of the involved helices.

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.

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.

Receptor oligomerization in family B1 of G-protein-coupled receptors: focus on BRET investigations and the link between GPCR oligomerization and binding cooperativity.

The superfamily of the seven transmembrane G-protein-coupled receptors (7TM/GPCRs) is the largest family of membrane-associated receptors. GPCRs are involved in the pathophysiology of numerous human diseases, and they constitute an estimated 30–40% of all drug targets. During the last two decades, GPCR oligomerization has been extensively studied using methods like bioluminescence resonance energy transfer (BRET) and today, receptor–receptor interactions within the GPCR superfamily is a well-established phenomenon. Evidence of the impact of GPCR oligomerization on, e.g., ligand binding, receptor expression, and signal transduction indicates the physiological and pharmacological importance of these receptor interactions. In contrast to the larger and more thoroughly studied GPCR subfamilies A and C, the B1 subfamily is small and comprises only 15 members, including, e.g., the secretin receptor, the glucagon receptor, and the receptors for parathyroid hormone (PTHR1 and PTHR2). The dysregulation of several family B1 receptors is involved in diseases, such as diabetes, chronic inflammation, and osteoporosis which underlines the pathophysiological importance of this GPCR subfamily. In spite of this, investigation of family B1 receptor oligomerization and especially its pharmacological importance is still at an early stage. Even though GPCR oligomerization is a well-established phenomenon, there is a need for more investigations providing a direct link between these interactions and receptor functionality in family B1 GPCRs. One example of the functional effects of GPCR oligomerization is the facilitation of allosterism including cooperativity in ligand binding to GPCRs. Here, we review the currently available data on family B1 GPCR homo- and heteromerization, mainly based on BRET investigations. Furthermore, we cover the functional influence of oligomerization on ligand binding as well as the link between oligomerization and binding cooperativity.

A BRET assay for monitoring insulin receptor interactions and ligand pharmacology

The insulin receptor (IR) belongs to the receptor tyrosine kinase super family and plays an important role in glucose homeostasis. The receptor interacts with several large docking proteins that mediate signaling from the receptor, including the insulin receptor substrate (IRS) family and Src homology-2-containing proteins (Src). Here, we applied the bioluminescence resonance energy transfer 2 (BRET2) technique to study the IR signaling pathways. The interaction between the IR and the substrates IRS1, IRS4 and Shc was examined in response to ligands with different signaling properties. The association between IR and the interacting partners could successfully be monitored when co-expressing green fluorescent protein 2 (GFP2) tagged substrates with Renilla reniformis luciferase 8 (Rluc8) tagged IR. Through additional optimization steps, we developed a stable and flexible BRET2 assay for monitoring the interactions between the IR and its substrates. Furthermore, the insulin analogue X10 was characterized in the BRET2 assay and was found to be 10 times more potent with respect to IRS1, IRS4 and Shc recruitment compared to human insulin. This study demonstrates that the BRET2 technique can be applied to study IR signaling pathways, and that this assay can be used as a platform for screening and characterization of IR ligands.

Beta-arrestin2 as a competitor for GRK2 interaction with the GLP-1 receptor upon receptor activation.

The signaling of seven transmembrane receptors/G-protein- coupled receptors (GPCRs) is regulated by a number of receptor interacting proteins, including βarrestins (βarrs) and GPCR kinases (GRKs). In the present report, we have analyzed the interaction pattern between the glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), βarr2, and GRK2 using bioluminescence resonance energy transfer assays. We found that βarr2 interacts with the GLP-1R in a biphasic manner with a phosphorylation-independent and a phosphorylation-dependent component. In competition experiments, we observed βarr2 competing with GRK2 for interaction with GLP-1R. We propose a model were βarr2 competes with GRK2 for interaction with the activated and GRK phosphorylated GLP-1R, suggesting a new role of βarr2 in regulating the orchestration of GRK2 functionality.

Improving membrane binding as a design strategy for amphipathic peptide hormones: 2‐helix variants of PYY3‐36

It has been hypothesized that amphipathic peptides might bind to membranes prior to activating their cognate receptors, but this has proven difficult to test. The peptide hormone PYY3‐36 is believed to perform its appetite‐suppressing actions through binding to hypothalamic Y2 receptors. It has been proposed that PYY3‐36 via its amphipathic α‐helix binds to the plasma membrane prior to receptor docking. Here, our aim was to study the implication of this hypothesis using new analogs of PYY3‐36. We first studied membrane binding of PYY3‐36. Next, we designed a series of PYY3‐36 analogs to increase membrane‐binding affinity by substituting the N‐terminal segment with a de novo designed α‐helical, amphipathic sequence. These 2‐helix variants of PYY3‐36 were assembled by solid‐phase peptide synthesis. Pharmacological studies demonstrated that even though the native peptide sequence was radically changed, highly active Y2 receptor agonists were generated. A potent analog, with a Kd of 4 nM for membranes, was structurally characterized by NMR in the membrane‐bound state, which clearly showed that it formed the expected 2‐helix. The topology of the peptide–micelle association was studied by paramagnetic relaxation enhancement using a spin label, which confirmed that the hydrophobic residues bound to the membrane. Our studies further support the hypothesis that PYY3‐36 associates with the membrane and indicate that this can be used in the design of novel molecules with high receptor binding potency. These observations are likely to be generally important for peptide hormones and biopharmaceutical drugs derived from them. This new 2‐helix variant of PYY3‐36 will be useful as a tool compound for studying peptide–membrane interactions. Copyright

Direct demonstration of NCAM cis‐dimerization and inhibitory effect of palmitoylation using the BRET2 technique

MINT‐8071483: NCAM140 (uniprotkb:P13591‐1) physically interacts (MI:0915) with NCAM140 (uniprotkb:P13591‐1) by competition binding (MI:0405)

A bioluminescence resonance energy transfer 2 (BRET2) assay for monitoring seven transmembrane receptor and insulin receptor crosstalk

Abstract The angiotensin AT1 receptor is a seven transmembrane (7TM) receptor, which mediates the regulation of blood pressure. Activation of angiotensin AT1 receptor may lead to impaired insulin signaling indicating crosstalk between angiotensin AT1 receptor and insulin receptor signaling pathways. To elucidate the molecular mechanisms behind this crosstalk, we applied the BRET2 technique to monitor the effect of angiotensin II on the interaction between Rluc8 tagged insulin receptor and GFP2 tagged insulin receptor substrates 1, 4, 5 (IRS1, IRS4, IRS5) and Src homology 2 domain-containing protein (Shc). We demonstrate that angiotensin II reduces the interaction between insulin receptor and IRS1 and IRS4, respectively, while the interaction with Shc is unaffected, and this effect is dependent on Gαq activation. Activation of other Gαq-coupled 7TM receptors led to a similar reduction in insulin receptor and IRS4 interactions whereas Gαs- and Gαi-coupled 7TM receptors had no effect. Furthermore, we used a panel of kinase inhibitors to show that angiotensin II engages different pathways when regulating insulin receptor interactions with IRS1 and IRS4. Angiotensin II inhibited the interaction between insulin receptor and IRS1 through activation of ERK1/2, while the interaction between insulin receptor and IRS4 was partially inhibited through protein kinase C dependent mechanisms. We conclude that the crosstalk between angiotensin AT1 receptor and insulin receptor signaling shows a high degree of specificity, and involves Gαq protein, and activation of distinct kinases. Thus, the BRET2 technique can be used as a platform for studying molecular mechanisms of crosstalk between insulin receptor and 7TM receptors.