Differential Responses of the GLP-1 and GLP-2 Receptors to N-Terminal Modification of a Dual Agonist

Abstract

Class B1 G protein-coupled receptors (GPCRs), collectively, respond to a diverse repertoire of extracellular polypeptide agonists and transmit the encoded messages to cytosolic partners. To fulfill these tasks, these highly mobile receptors must interconvert among conformational states in response to agonists. We recently showed that conformational mobility in polypeptide agonists themselves plays a role in activation of one class B1 GPCR, the receptor for glucagon-like peptide-1 (GLP-1). Exchange between helical and nonhelical conformations near the N-termini of agonists bound to the GLP-1R was revealed to be critical for receptor activation. Here, we ask whether agonist conformational mobility plays a role in the activation of a related receptor, the GLP-2R. Using variants of the hormone GLP-2 and the designed clinical agonist glepaglutide (GLE), we find that the GLP-2R is quite tolerant of variations in α-helical propensity near the agonist N-terminus, which contrasts with signaling at the GLP-1R. A fully α-helical conformation of the bound agonist may be sufficient for GLP-2R signal transduction. GLE is a GLP-2R/GLP-1R dual agonist, and the GLE system therefore enables direct comparison of the responses of these two GPCRs to a single set of agonist variants. This comparison supports the conclusion that the GLP-1R and GLP-2R differ in their response to variations in helical propensity near the agonist N-terminus. The data offer a basis for development of new hormone analogues with distinctive and potentially useful activity profiles; for example, one of the GLE analogues is a potent agonist of the GLP-2R but also a potent antagonist of the GLP-1R, a novel form of polypharmacology.

Figure 1.

Sequences of glepaglutide (GLE), GLP-2, GLP-1, and Ex-4. The residues of GLE are color-coded based on parallels to residues found in GLP-2, GLP-1 or Ex-4 at matched positions. Values in brackets indicate the number of shared residues.

Figure 2.

Structures of amino acid residues used to replace Gly4 of GLP-2-NH₂ or GLE in this study. Left: L-Alanine (L-Ala) and cyclic β-amino acid residue (S,S)-trans-aminocyclopentanecarboxylic acid ((S,S)-ACPC) are helix-stabilizing replacements. Right: D-Alanine (D-Ala), (R,R)-ACPC) and β-homoglycine (β-hGly; also called β-alanine) are helix-destabilizing replacements.

Figure 3.

nLuc-GLP-2R competition binding assay for measuring the affinity of GLP-2, GLE and their derivatives. (a) Cartoon depiction of the nLuc-GLP-2R bioluminescence resonance energy transfer (BRET) assay. (b) The effect of nanoluciferase (nLuc) fusion to the GLP-2R was evaluated in HEK293 cells stably expressing the GloSensor^™ protein. cAMP production was measured in response to GLP-2 or GLE. Data points represent the mean of ≥2 independent experiments. (c) Production of cAMP in response to GLP-2 or GLE was assessed in HEK293 cells expressing the native GLP-2R and GloSensor^™ proteins. Data points represent the mean of ≥2 independent experiments. In both (b) and (c), data were normalized relative to GLP-2; errors bars represent S.E.M.

Figure 4.

The effects of Gly4 replacement on GLP-2R activation and nLuc-GLP-2R binding by GLP-2 and GLP-2-NH₂ analogues. (a) Sequences of GLP-2 and GLP-2-NH₂ analogues with substitutions at Gly4. (b) Activation of the GLP-2R by GLP-2 and GLP-2-NH₂ analogues, as measured by cAMP production in GloSensor^™ HEK293 cells transiently expressing the GLP-2R. Data points represent the average of ≥3 independent experiments. Data were normalized relative to GLP-2. (c) nLuc-GLP-2R competition binding, as measured by bioluminescence resonance energy transfer, for unlabeled GLP-2 and GLP-2-NH₂ analogues. Assays were performed in GloSensor^™ HEK293 cells transiently expressing the nLuc-GLP-2R. 20 nM of tetramethylrhodamine (TMR)-labeled GLP-2(1-34)-(Lys34-TMR)-NH₂ tracer was used. Data points represent the mean of ≥4 independent experiments. All uncertainties in (b) and (c) are expressed as S.E.M.

Figure 5.

The effects of Gly4 replacement on GLP-2R activation, nLuc-GLP-2R binding, GLP-1R activation and nLuc-GLP-1R binding by GLE and analogues. (a) Sequences of GLE and analogues with substitutions at Gly4. (b) Activation of the GLP-2R by GLE and analogues as measured by cAMP production in HEK293 cells transiently expressing the GLP-2R and stably expressing the GloSensor^™. Data points represent the average of ≥4 independent experiments. (c) nLuc-GLP-2R competition binding, as measured by bioluminescence resonance energy transfer (BRET), for unlabeled GLE and analogues. Assays were performed in GloSensor^™ HEK293 cells transiently expressing the nLuc-GLP-2R. 20 nM of tetramethylrhodamine (TMR)-labeled GLP-2(1-34)-(Lys34-TMR)-NH₂ tracer was used. Data points represent the mean of ≥5 independent experiments. (d) GLP-1R activation, as measured by cAMP production in GloSensor^™ HEK293 cells transiently expressing the GLP-1R. Data points represent the average of ≥3 independent experiments. (e) nLuc-GLP-1R competition binding, as measured by BRET, for unlabeled GLE and analogues. Assays were performed in GloSensor^™ HEK293 cells transiently expressing the nLuc-GLP-1R. 5 nM of tetramethylrhodamine (TMR)-labeled GLP-1(7-36)-(Lys18-TMR)-NH₂ tracer was used. Data points represent the mean of ≥3 independent experiments. Data in (b) and (d) were normalized relative to GLE. All uncertainties in (a-d) are expressed as S.E.M.

Figure 6.

Inhibition of GLP-1(7-36)-stimulated cAMP production at the GLP-1R by GLE analogues. (a) Sequences of Ex(9-39), a previously reported GLP-1R antagonist, and GLE analogues modified at Gly4. (b) Inhibition of cAMP production after pretreating GloSensor^™ HEK293 cells transiently expressing the GLP-1R with the indicated GLE analogues for 30 min followed by stimulation with 0.25 nM GLP-1. Data points represent the average of ≥4 independent experiments. All error bars represent S.E.M.