Glucagon-Like Peptide-1 Receptor Dimerization Differentially Regulates Agonist Signaling but Does Not Affect Small Molecule Allostery

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Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

Kaleeckal G. Harikumara,1, Denise Woottenb,1, Delia I. Pinona, Cassandra Kooleb,AlicjaM.Balla, Sebastian G. B. Furnessb, Bim Grahamc, Maoqing Donga, Arthur Christopoulosb, Laurence J. Millera,2, and Patrick M. Sextonb,2

aDepartment of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ 85259; and bDrug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, and cMedicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia

Edited* by Brian K. Kobilka, Stanford University School of Medicine, Stanford, CA, and approved September 24, 2012 (received for review March 28, 2012)

The glucagon-like peptide-1 receptor (GLP-1R) is a family B G protein- (17–25). There is also emerging evidence for functionally significant coupled receptor and an important drug target for the treatment of heterodimerization (25–27). Furthermore, although there is an fi type II diabetes, with activation of pancreatic GLP-1Rs eliciting emerging theme in which dimerization contributes to high-af nity glucose-dependent insulin secretion. Currently, approved therapeu- peptide binding and cAMP signaling (17, 18), how dimerization tics acting at this receptor are peptide based, and there is substantial contributes more globally to receptor signaling and whether it plays a role in ligand-directed stimulus bias are unknown. interest in small molecule modulators for the GLP-1R. Using a variety There is parallel interest in the development of allosteric drugs of resonance energy transfer techniques, we demonstrate that the targeting otherwise intractable GPCRs to enable enhanced selec- GLP-1R forms homodimers and that transmembrane helix 4 (TM4) tivity, fine control of receptor function, and/or maintenance of provides the primary dimerization interface. We show that disruption spatial and temporal elements of endogenous (orthosteric) signal- of dimerization using a TM4 peptide, a minigene construct encoding ing (1, 28). However, little is known on whether such drugs act TM4, or by mutation of TM4, eliminates G protein-dependent high- through cooccupancy of a single receptor protomer (in cis), or affinity binding to GLP-1(7-36)NH2 but has selective effects on recep- asymmetrically across dimers (in trans), with most drug develop-

tor signaling. There was <10-fold decrease in potency in cAMP accu- ment assumptive on an in cis mechanism of action. Allosteric drugs PHARMACOLOGY mulation or ERK1/2 phosphorylation assays but marked loss of are likely to be required for targeting of family B GPCRs, given the intracellular calcium mobilization by peptide agonists. In contrast, diffuse orthosteric pharmacophore of their peptide agonists (29) fi there was near-complete abrogation of the cAMP response to an and the dif culty in mimicking this with small molecule compounds. allosteric agonist, compound 2, but preservation of ERK phosphory- Understanding how such molecules bind and modulate receptor lation. Collectively, this indicates that GLP-1R dimerization is impor- function is crucial for successful optimization of such drugs. In this study, we demonstrate functionally important homo- tant for control of signal bias. Furthermore, we reveal that two small dimerization of the GLP-1 receptor (GLP-1R) that occurs through molecule ligands are unaltered in their ability to allosterically modu- an interface along transmembrane segment 4 (TM4) and that this late signaling from peptide ligands, demonstrating that these mod- dimerization is critical for selective coupling of the receptor to ulators act in cis within a single receptor protomer, and this has physiologically relevant signaling pathways. Furthermore, dimer- important implications for small molecule drug design. ization is important for the signal bias of the receptor and dis- criminates between peptide and nonpeptide-mediated receptor allosteric modulation | biased signaling | G protein-coupled receptors | activation, but is not required for small molecule allosteric mod- family B GPCRs ulation of the receptor, indicating that rational design of allosteric therapeutics is possible within a single receptor protomer model. protein-coupled receptors (GPCRs) are the largest super- Results Gfamily of cell surface proteins and play crucial roles in virtually every physiological process. Their widespread abundance, yet se- GLP-1 Receptor Forms Functionally Important Homodimers. A num- lective distribution, and ability to couple to a variety of signaling ber of complementary biochemical, biophysical, and pharmacological approaches were used to demonstrate the oligomeric and effector systems make them extremely attractive targets for – drug development (1). Recently, there has been increasing interest nature of GLP-1R interactions and the impact on ligand receptor interactions. As previously observed with other family B GPCRs in the stoichiometry of receptors involved in GPCR signaling – complexes and how this may impact on receptor function and drug (17, 18, 25), analysis of receptor receptor interaction in live cells discovery (2–4). using a combination of static and saturation bioluminescence With the exception of family C GPCRs, where obligate di- resonance energy transfer (BRET) analyses revealed constitutive, specific homooligomerization of the GLP-1R (Fig. 1 A and B). No merization can occur (4), the role of oligomerization in GPCR fi function has remained controversial (2–8), and this has been the speci c BRET signal was seen with coexpression of the BRET subject of a number of recent reviews (9–11). Although there is an donor GLP-1R-Rluc and the family A cholecystokinin 2 GPCR increasing body of evidence supporting dimerization of GPCRs (CCK2R), or between GLP-1R-YFP and soluble Rluc protein, as a widespread feature of GPCR biology, including numerous studies on family A GPCRs, whether these are stable, transient, constitutive, or ligand dependent, and how they impact on Author contributions: K.G.H., D.W., M.D., A.C., L.J.M., and P.M.S. designed research; K.G.H., receptor function and drug discovery are less clear, and general D.W., D.I.P., C.K., A.M.B., S.G.B.F., B.G., and M.D. performed research; B.G. contributed rules for oligomeric behavior are not evident (9–16). Even where new reagents/analytic tools; K.G.H., D.W., C.K., A.C., L.J.M., and P.M.S. analyzed data; and K.G.H., D.W., S.G.B.F., A.C., L.J.M., and P.M.S. wrote the paper. effects on signaling are studied, these are generally linked to a single pathway and the role of dimerization in the control of The authors declare no conflict of interest. receptor engagement and preference for distinct intracellular *This Direct Submission article had a prearranged editor. signaling intermediates (i.e., signal bias) is virtually unstudied. 1K.G.H. and D.W. contributed equally to this work. For family B GPCRs, which encompass many therapeutically 2To whom correspondence may be addressed. E-mail: [email protected] or patrick.sexton@ important peptide receptors, including those for glucagon, gluca- monash.edu. gon-like peptides 1 and 2 (GLP-1, GLP-2), parathyroid hormone, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and calcitonin, there is consistent evidence for homodimerization 1073/pnas.1205227109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1205227109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 although a strong BRET signal was observed for the CCK2R the mutant receptors was not due to changes in cell surface homodimer (Fig. S1 A and B), demonstrating specificity of the expression because whole-cell binding measurements revealed that GLP-1R homooligomeric interaction. This interaction was also expression of these mutant receptors was not significantly different not significantly altered by saturating concentrations of either from the wild-type receptor. Analysis of GLP-1(7-36)NH2 peptide – peptide [GLP-1(7-36)NH2, exendin-4, oxyntomodulin] or non- inhibition binding, in GLP-1R expressing Cos-1 membranes, peptide (6,7-dichloro2-methylsulfonyl-3-tert-butylaminoquinoxa- revealed a complex pattern of binding that was best fit with a three- line, compound 2) (30) agonists of the receptor (Fig. S1C). site mass action model (Fig. 1E, Table 1). Disruption of di- We also used a combination of bimolecular-fluorescence com- merization via the TM4 mutants led to complete loss of the very- plementation and BRET to probe whether evidence for higher high-affinity state (K1) and marked reduction in the proportion of order oligomers of the GLP-1R could be established. However, high-affinity (K2) sites (Table 1, Fig. 1F, Fig. S5A), and a similar coexpression of GLP-1R-Rluc with both the GLP-1R-YN and effect is observed with coincubation of the wild-type receptor with GLP-1R-YC constructs failed to yield a significant BRET signal the TM4 peptide (Fig. S5B). This effect was mimicked by in- (Fig. S1E), despite the generation of functional YFP from cubation of membranes with the nonhydrolyzable GTP analog, the fluorescence complementation established at the level of the GppNHp (Fig. 1E, Table 1), suggesting that the principal effect of dimeric receptor complex (Fig. S1D). All GLP-1R fusion con- dimerization is to maintain a high-affinity G protein-complexed structs had equivalent binding and cAMP signaling to the wild- state. Coincubation of GLP-1R TM mutant membranes with type receptor (Fig. S2, Table S1). These data indicate that the GppNHp had minimal additional effect on agonist peptide binding GLP-1R dimer is the major oligomeric form of the receptor. (Table 1, Fig. 1F), whereas the negative cooperativity observed in Negative cooperativity of peptide binding to the secretin family B dissociation kinetic studies (Fig. S3A) was abolished in the G252A, GPCR has been described (31), and this was also observed in L256A, V259A GLP-1R mutant (Fig. S3B). dissociation kinetic studies of the GLP-1R (Fig. S3A), confirming the oligomeric nature of the GLP-1R. Dimerization Plays Distinct Roles in GLP-1R Peptide and Small As seen with the related calcitonin and secretin family B GPCRs Molecule Agonist Signaling. To date, investigation of the role of (17, 18), disruption of the TM4 interface by either GLP-1R homodimerization on family B receptor signaling has been con- TM4 peptides or by mutation of the hydrophobic face of TM4 fined to examination of cAMP formation (17, 18). Family B (L256A, V259A, or G252A, L256A, V259A; Fig. S4) abolished the receptors, however, are pleiotropically coupled, with multiple BRET signal (Fig. 1 A and B), consistent with TM4 constituting the signaling pathways important for receptor function (32). In addi- principal dimer interface for GLP-1Rs as well as other family B tion to cAMP formation, both phosphorylation of ERK and mo- GPCRs (17, 18, 31). Disruption of GLP-1R dimerization either bilization of intracellular calcium are physiologically important by coincubation of GLP-1R BRET constructs with GLP-1R TM4 in GLP-1–mediated control of pancreatic β-cell function (33). We (Fig. 1C) or mutation of TM4 (Fig. 1D), led to a ∼10-fold decrease have used the dimer-disrupting triple mutant (GLP-1R G252A, in GLP-1(7-36)NH2 potency to form cAMP, indicating that L256A, V259A) to probe the extent to which dimerization con- dimerization is important for efficient coupling of the receptor to tributes to GLP-1R signaling across these three pathways. A sim- the Gs/AC/cAMP signaling cascade. The decrease in potency at ilar (<10-fold) decrease in GLP-1(7-36)NH2 potency was observed

Fig. 1. Dimerization of the GLP-1R is important for high-afﬁnity agonist binding and receptor coupling to cAMP production. Saturation (A) and static (B) BRET data from transient coexpression, in Cos-1 cells, of Rluc- and YFP-tagged GLP-1R constructs of wild- type, in the presence or absence of TM4 peptides from the GLP-1R or from the secretin receptor, or dimer disrupting mutants of TM4. In the presence of the GLP-1R TM4 peptide or when TM4 double/triple mutants were present, the static BRET ratio was re- duced and was not signiﬁcantly different from back-

ground signal. (C) GLP-1(7-36)NH2 stimulated cAMP responses in CHO cells stably expressing the GLP-1R in the presence or absence of the GLP-1 receptor TM4

segment peptide. (D) GLP-1(7-36)NH2 stimulated cAMP responses in Cos-1 cells transiently expressing either the wild-type or L256A, V259A, or G252A, L256A, V259A TM4 mutant GLP-1Rs. The cell surface expression of the G252A, L256A, V259A mutant was 112 ± 10% of the wild-type receptor. (E) Inhibition of 125 I-GLP-1(7-36)NH2 binding, to membranes from Cos- 1 cells transiently transfected with the wild-type GLP-

1R, by unlabeled GLP-1(7-36)NH2 in the presence or absence of 10 μM GppNHp. (F) Inhibition of 125I-GLP-1

(7-36)NH2 by unlabeled GLP-1(7-36)NH2 in the presence or absence of 10 μM GppNHp, to membranes from Cos-1 cells transiently transfected with the G252A, L256A, V259A mutant GLP-1R. For reference, the curve for the WT receptor is shown with a dashed line. Values are means ± SEM of data from four to ﬁve independent experiments performed in duplicate.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1205227109 Harikumar et al. Downloaded by guest on September 28, 2021 Table 1. Disruption of GLP-1R dimerization attenuates high- Discussion affinity GLP-1 binding The occurrence and significance of dimerization remains con- Receptor construct K K K troversial outside of family C GPCRs, where there is evidence 1 2 3 for obligate oligomerization. For family B receptors, there is Binding affinity (pIC50)* 10.29 ± 0.60 8.40 ± 0.13 6.99 ± 0.12 consistent evidence for homooligomerization, together with the Fraction potential for functionally important heterooligomerization (18– −GppNHp 23, 25–27). Within this theme, there is an emerging paradigm of GLP-1R 0.14 ± 0.06 0.76 ± 0.08 0.10 behavior in which homodimerization of receptors occurs via a GLP-1R(L256A, V259A) 0.00 ± 0.04 0.46 ± 0.08 0.54 TM4/TM4 interface and that this interaction is required for fi GLP-1R(G252A, 0.00 ± 0.03 0.31 ± 0.08 0.69 optimal function of the receptor, generation of high-af nity ag- L256A, V259A) onist binding, and signaling via formation of cAMP (17, 18, 31); +GppNHp this paradigm is true also for the GLP-1R. A major finding of the current study is that disruption to the GLP-1R 0.00 ± 0.05 0.38 ± 0.09 0.62 ± ± dimer interface had differential impact on signaling engaged by GLP-1R(G252A, 0.00 0.06 0.14 0.10 0.86 the GLP-1R, with attenuation of cAMP formation and phos- L256A, V259A) phorylation of ERK, but almost complete abrogation of the in- Binding data were analyzed with a three-site logistic fit and presented tracellular calcium mobilization response. Although differential as –logM estimates. effects on the signal pathways could be due to different strength *Binding affinity parameters were shared across the data set. of stimulus coupling, these results cannot be interpreted as simply a consequence of strength of stimulus coupling as pERK1/2 (in addition to intracellular calcium mobilization) is also less well in cAMP accumulation and ERK phosphorylation assays (Fig. 2 A coupled than cAMP and this pathway is only minimally (less so and B), with no change in the time course for pERK (Fig. S6). than cAMP) affected by disruption of dimerization. This suggests However, there was almost complete abrogation of the in- that fine control of GLP-1R signaling, and the ability to engage tracellular calcium response (Fig. 2C), demonstrating distinct with its full range of signaling intermediates is linked to the ability consequences of dimerization across pathways. Oxyntomodulin is of the receptor to form dimers. As receptor-mediated intracel- an endogenous agonist of the GLP-1R that exhibits signal bias lular calcium mobilization is less well coupled than Gs-mediated relative to that of GLP-1 peptides for at least cAMP versus pERK cAMP formation, it may require both receptor dimerization and (34), whereas exendin-4 is clinically used as a GLP-1 mimetic (35). occupancy of both receptor protomers within the dimer to elicit Disruption of dimerization had similar effect on the responses to the response, providing a mechanism through which receptors PHARMACOLOGY all three peptides (Fig. 2 D and E, Fig. S7), with loss of calcium and ligands achieve the conformational complexes associated − signaling maintained even at 10 5 M. In contrast, there was with signal bias of the receptor. However, this is not the case for marked loss of cAMP signaling by the small molecule agonist, coupling to ERK1/2 phosphorylation. The GLP-1R has been compound 2, but only a relatively small reduction in pERK sig- shown to signal downstream of coupling to Gs, Gi/o, and Gq naling (Fig. 2 G and H), the latter likely because of decreased proteins as well as β-arrestins (33, 37), with at least part of the affinity of that compound (Fig. 3). Equivalent effects were seen in intracellular calcium signal being Gi dependent (38) and at least CHO-FlpIn cells transfected with the mutant receptors (Fig. S8), part of the pERK response dependent on β-arrestin (37). It is demonstrating that the effects were independent of cellular likely that the disruption to dimerization seen in the current study background. To further confirm that the differential effects on differentially alters the coupling efficiency of the receptor to these signaling were due to disruption of dimerization, we have also different effectors. Furthermore, it provides a potential mecha- used a minigene encoding TMs 3 and 4 of the GLP-1R, with TM3 nistic basis through which changes to G protein-coupling pref- required to enable correct orientation of the TM4 segment. erences occur at higher concentrations of agonist. Transfection of this construct into CHOflpIn cells stably A second major finding of the current study was the distinct expressing the GLP-1R yielded qualitatively similar results to the contribution of dimerization to orthosteric versus allosteric ac- mutant receptor (Fig. S9), although the magnitude of effect for all tivation of cAMP signaling through the GLP-1R, with effective three pathways was dependent on transfection efficiency. Col- abrogation of cAMP response (>30-fold) to compound 2, lectively, these data indicate distinct modes of receptor activation whereas there was only limited (<8-fold) loss of peptide-medi- for peptide versus small molecule agonists of the GLP-1R. The ated cAMP formation. This implies that compound 2 has limited differential effects on signaling pathways is unlikely to be due to capacity to activate Gs protein via monomeric GLP-1R and that simply changing coupling efficiency across the board, because the the allosteric agonist has a distinct mode of receptor activation relative potency of ligands for pERK and calcium signaling are to the peptide agonists. This further supports a role for di- similar at the wild-type receptor, with dramatically larger effects merization in controlling signal bias (rather than a phenomenon of disrupting dimerization on calcium mobilization. related to strength of coupling) as disruption of dimerization has a stronger effect on cAMP formation than on phosphorylation Allosteric Modulation of the GLP-1R Occurs Within a Single Receptor of ERK, despite greater relative efficacy for compound 2 in Protomer. Currently, very little is known with respect to how allo- formation of cAMP. A differential mode of receptor activation steric modulators exert their cooperativity, including whether this by compound 2 is consistent with findings of distinct effects of requires dimerization of receptors or is due to coincident binding to receptor polymorphism (T149M) or mutation (ECL2 mutants) a single monomeric unit. As demonstrated previously (34), com- on peptide versus allosteric agonist activation of the receptor, pound 2 displays weak positive cooperativity with GLP-1(7-36)NH2 where the allosteric agonism is preserved at receptors with impaired and strong positive cooperativity with oxyntomodulin (Fig. 3 A and peptide response (38, 39). Furthermore, it demonstrates that di- C, Fig. S10, and Table 2). This cooperativity was not significantly merization can contribute to ligand-directed signal bias. altered by the dimer-disrupting mutant, despite the loss of agonism A third significant finding of the current study was that allo- seen with compound 2 (Fig. 3 B and D, Fig. S10, and Table 2). An steric modulation of orthosteric peptide binding and signaling equivalent preservation of allosteric cooperativity was seen with the was maintained after disruption of the dimer, despite loss of structurally distinct modulator, 4-(3-benzyloxy-phenyl)-2-ethyl- allosteric agonism, demonstrating that the allosteric modulation sulfinyl-6(trifluoromethyl)pyrimidine (BETP) (Fig. 3 E and F). To was occurring in cis within a single protomer. The use of two confirm that this cooperativity was not probe dependent, we also chemically distinct modulators, compound 2 from NovoNordisk examined the allosteric modulation of the GLP-1 metabolite, GLP- (30) and BETP from Eli Lilly (40), confirmed that this is likely to 1(9-36)NH2 (Fig. S11) (36). These data demonstrate that the be a generalized feature of allosteric modulation of the GLP-1R. allosteric modulation of the GLP-1R occurs within a single Understanding how allosteric drugs alter the function of orthos- monomeric unit and does not require dimerization of the receptor. teric ligands is important for drug development, particularly for

Harikumar et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 Fig. 2. Disruption of GLP-1R dimerization differentially modiﬁes signal bias and orthosteric versus allosteric agonist function. Concentration–response curves

were generated for GLP-1(7-36)NH2 (A–C), oxyntomodulin (D–F), and compound 2 (G and H) in three different functional assays, cAMP accumulation (A, D, and G), ERK1/2 phosphorylation (B, E, H), and calcium mobilization (C and F) using cells transiently transfected with either wild-type GLP-1R or G252A, L256A, V259A GLP-1R. Cell surface expression of the mutant was not significantly different from WT (112 ± 10%). cAMP and pERK data are from transiently transfected Cos-1 cells and calcium mobilization data from transfected CHOflpIn cells due to the limited calcium response in Cos-1 cells. Comparative data for all pathways and all peptide ligands in CHOflpIn cells are shown in Fig. S8. Data are normalized to maximal peptide response observed at the wild-type receptor. Data points represent the mean ± SEM of four individual experiments performed in duplicate.

rational design of ligands in which exclusion from the orthosteric versus dimeric receptors in the membrane has been difficult to binding site of a single receptor protomer is generally assumed. study, and this is not known for any family B GPCR. Recent The base paradigm illustrated in the current study, that of work with family A receptors suggests that many GPCRs exist in critical importance of dimerization for efficient coupling to Gs/ a dynamic equilibrium between monomeric and dimeric states, AC/cAMP, maintenance of very high-affinity agonist peptide with the stability of dimeric complexes differing for individual binding (presumably linked to occupancy of a single protomer in receptor subtypes (12–15). Nonetheless, even for highly transient the dimer), and negative cooperativity between agonist-occupied interactions, such as those seen for FPR receptors, the mean receptors is similar to that described for the dopamine D2 re- residence time for interaction (∼90 ms) (15) would allow for ceptor, in which optimal physiological signaling is predicted to coupling to and activation of G proteins (40-70 ms) (44, 45), occur through activation of a single protomer within the dimeric consistent with dimerization playing an important role in the complex (7). Interestingly, although multiple domains (TM4/5 efficiency of G-protein coupling and, indeed, in differentiating and TM1) of the D2 receptor have been implicated in oligo- strength of stimulus and pleiotropic receptor coupling. merization (41–43), there is an activation-related conformational In conclusion, our work provides mechanistic insight into the change that occurs via the TM4 dimer interface (42). With the role of the oligomeric status of the GLP-1R for biased agonism current study, a functionally important TM4 dimeric interface and allosteric modulation of the receptor, and this has important has now been demonstrated in the three family B GPCRs that implications for drug discovery and development at the thera- have been studied to date (17, 18), and may imply a more general peutically attractive B family of GPCRs. role for allosteric conformational transition across receptor protomers for this TM domain. In this vein, it is interesting to note Methods that heterodimerization of GLP-1 and GIP receptors led to al- GLP-1R Constructs. Human GLP-1R constructs taggedwith Renilla luciferase (Rlu) tered GLP-1–induced arrestin recruitment and intracellular or yellow fluorescent protein (YFP) inserted in frame at the carboxyl terminus of calcium mobilization (27), and although the dimer interface for the mature protein were prepared, as described previously (26). GLP-1R alanine- family B GPCR heterodimers has yet to be empirically estab- replacement mutants were generated using the QuikChange site-directed lished, it is possible that allosteric regulation of receptors across mutagenesis kit (Stratagene), replacing residues 252, 256, and 259 within the TM4 is relevant for signaling even for heteromeric complexes. predicted lipid-exposed face of TM4 separately and in combination. Additional It is unclear whether the TM4 peptide or the TM4 mutation GLP-1R constructs were prepared by inserting amino-terminal or carboxyl-ter- fully abrogates dimerization or changes the interaction from minal portions of YFP, representing residues 1–158 (YN) or residues 159–238 stable to transient, such that a BRET signal is not observed. (YC) just before the TGA stop codon. The sequences of all receptor constructs Nonetheless, it is clear that stable dimerization is required for were verified by direct DNA sequencing. All constructs had equivalent binding critical elements of GLP-1R function. The content of monomeric affinity and potency in cAMP accumulation assays (Table S1, Fig. S2).

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Fig. 3. Allosteric modulation of the GLP-1R occurs within a single receptor protomer. Concentration–response curves

were generated for GLP-1(7-36)NH2 (A, C, and E) and oxyntomodulin (B, D,and F) in the presence and absence of increasing concentrations of compound 2(A–D)orBETP(E and F)incAMP accumulation assays using Cos-1 cells expressing wild-type GLP-1R (Left)or G252A, L256A, V259A GLP-1R (Right). Data are normalized to maximal peptide response, are ﬁtted with an operational model of allosterism, and are representa- tive of the mean ± SEM of four independent experiments performed in duplicate.

Transfections and Cell Culture. Cos-1, CHO, or FlpInCHO cells used for tran- metafectene (Invitrogen) following the manufacturer’s protocol. CHO cells sient transfections were maintained in DMEM supplemented with 5% stably expressing the human GLP-1 receptor (CHO-GLP-1R) were propagated (vol/vol) heat-inactivated FBS and incubated in a humidiﬁed environment in Ham’s F-12 medium supplemented with 5% (vol/vol) FBS (46). For all

at 37 °C in 5% (vol/vol) CO2. GLP-1R receptor constructs were transiently whole-cell assays, cells were seeded into 96-well culture plates 24 h after transfected using either the DEAE-dextran method as previously described or transfection at a density of 15,000–20,000 cells/well for Cos-1 cells or 30,000

Table 2. Allosteric parameters for WT and G252A, L256A, V259A GLP-1Rs GLP-1R wild type GLP-1R(G252A, L256A, V259A)

Interacting ligands pKB Logαβ (αβ)pKB Logαβ (αβ)

Compound 2 5.56 ± 0.13 4.71 ± 0.21

GLP-1(7-36)NH2 0.59 ± 0.11 (3.9) 0.13 ± 0.15 (1.3) Oxyntomodulin 1.46 ± 0.15 (29) 1.74 ± 0.12 (55) BETP 5.20 ± 0.20 4.78 ± 0.15 Oxyntomodulin 1.14 ± 0.14 (14) 1.62 ± 0.11 (42)

Quantitative parameters for the allosteric interaction of compound 2 or BETP with GLP-1(7-36)NH2 or oxyntomodulin. cAMP data were analyzed with an operational model of allosterism as deﬁned in Methods.

pKB values represent the negative logarithm of the afﬁnity for the allosteric ligands derived from application of the operational model of allosterism. Logαβ values represent the composite cooperativity between the allosteric modulator and the orthosteric ligand. Antilogarithms are shown in parentheses.

Harikumar et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 cells/well for CHOFlpIn cells and incubated overnight at 37 °C in 5% (vol/vol) ERK1/2 Phosphorylation Assay. Receptor-mediated ERK1/2 phosphorylation

CO2 before assaying. The level of cell surface-expressed mutant and wild- was determined by using the AlphaScreen ERK1/2 SureFire protocol as de- type receptors was not significantly different. For robustness, experiments scribed previously (34). were performed in multiple cellular backgrounds, with a subset of control + + experiments performed in both the American and Australian laboratories. Intracellular Ca2 Mobilization Assay. Intracellular Ca2 mobilization was de- All signaling assays performed in Cos-1 cells transiently expressing the wild- termined as described previously (34). type or mutant receptors were repeated in transiently transfected CHOFlpIn cells (Fig. S8). Data Analysis. All data obtained were analyzed in GraphPad Prism 5.0.3 (GraphPad Software). Radioligand inhibition binding data were fitted to BRET Studies. BRET studies were performed on receptor-expressing Cos-1 cells a three-site inhibition mass action curve. In whole-cell ligand interaction as previously described (18). studies, data were fitted to an operational model of allosterism and agonism to derive functional estimates of modulator affinity and cooperativity. For all Bimolecular Fluorescence Complementation. Bimolecular fluorescence com- other data, concentration–response curves were fitted with a three-param- plementation assays were carried out in HEK293 cells expressing YN- and eter logistic equation. YC-tagged receptor constructs, as described previously (19). For more detailed methods, see SI Methods. cAMP Assays. cAMP accumulation assays were carried out using the AlphaScreen kit or the LANCE assay as previously described (18, 34). ACKNOWLEDGMENTS. We thank Mary-Lou Augustine for her excellent technical assistance and Drs. Kyle Sloop and Francis Willard for supplying BETP. P.M.S. is a Principal Research Fellow and A.C. is a Senior Research Fellow Radioligand Binding Assays. Receptor binding assays were carried out either of the National Health and Medical Research Council of Australia (NHMRC). using intact Cos-1 cells expressing the tagged receptor or isolated receptor- This work was funded in part by NHMRC Project Grant 1002180, NHMRC bearing membranes, as described previously (25, 34). Program Grant 519461, and National Institutes of Health Grant DK46577.

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