Transmembrane Segment IV Contributes a Functionally Important Interface for Oligomerization of the Class II G Protein-coupled Secretin Receptor

Abstract

Oligomerization of the Class II G protein-coupled secretin receptor has been reported, but the molecular basis for this and its functional significance have not been determined. In the current work, we have examined the possible contribution of each of the transmembrane (TM) segments of this receptor to its homo-oligomerization, using the method of competitive disruption screening for inhibition of receptor bioluminescence resonance energy transfer signal. TM IV was the only segment that was found to disrupt receptor bioluminescence resonance energy transfer. Evaluation of predicted interhelical and lipidexposed faces of this TM segment demonstrated that its lipidexposed face represented the determinant for oligomerization. This was further confirmed by mutagenesis of the intact secretin receptor. Morphological FRET was utilized to demonstrate that secretin receptor oligomerization occurred at the cell surface and that this oligomerization was disrupted by mutating Gly 243 and Ile 247 , key residues within the lipid-exposed face of TM IV. Although disruption of the receptor oligomerization interface had no effect on secretin binding parameters, it reduced the ability of secretin to stimulate intracellular cAMP. This supports a clear functional effect of oligomerization of this receptor. Such an effect might be particularly relevant to clinical situations in which this receptor is overexpressed, such as in certain neoplasms.Dimerization of plasma membrane receptors represents a timely theme of substantial interest and potential importance (1). For single transmembrane tyrosine kinase receptors, dimerization has been shown to provide a molecular mechanism for cross-molecular phosphorylation and receptor regulation (2). For some G protein-coupled receptors, dimerization or oligomerization has also been demonstrated, although the rules for establishment of such complexes, the effects of ligand binding to these complexes, and its functional implications have been quite varied (3-6). The role of such complexes in normal physiology is also unclear, with most demonstrations of G protein-coupled receptor oligomerization occurring in the setting of receptor overexpression systems (7,8) We previously demonstrated the oligomerization of the Class II G protein-coupled secretin receptor, based on a positive bioluminescence resonance energy transfer (BRET) 2 signal from tagged receptors expressed in COS cells that was structurally specific, not shared with similar levels of overexpression of a Class I G protein-coupled receptor with the secretin receptor (9). Subsequently, using the same technique and complementing this with morphologic fluorescence resonance energy transfer (FRET) analysis, the secretin receptor oligomerization was shown to occur constitutively at the cell surface, without being disrupted by secretin agonist ligand binding (10). Further, the secretin receptor was shown to be capable of forming similar hetero-oligomers with structurally related VPAC1 and VPAC2 receptors (10). Of interest, expression of a secre...

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

“…S4) abolished the BRET signal ( Fig. 1 A and B), consistent with TM4 constituting the principal dimer interface for GLP-1Rs as well as other family B GPCRs (17,18,31). Disruption of GLP-1R dimerization either by coincubation of GLP-1R BRET constructs with GLP-1R TM4 (Fig.…”

“…As previously observed with other family B GPCRs (17,18,25), analysis of receptor-receptor interaction in live cells using a combination of static and saturation bioluminescence resonance energy transfer (BRET) analyses revealed constitutive, specific homooligomerization of the GLP-1R (Fig.…”

“…As seen with the related calcitonin and secretin family B GPCRs (17,18), disruption of the TM4 interface by either GLP-1R TM4 peptides or by mutation of the hydrophobic face of TM4 (L256A, V259A, or G252A, L256A, V259A; Fig. S4) abolished the BRET signal ( Fig.…”

“…For family B GPCRs, which encompass many therapeutically important peptide receptors, including those for glucagon, glucagon-like peptides 1 and 2 (GLP-1, GLP-2), parathyroid hormone, and calcitonin, there is consistent evidence for homodimerization (17)(18)(19)(20)(21)(22)(23)(24)(25). There is also emerging evidence for functionally significant heterodimerization (25)(26)(27).…”

“…There is also emerging evidence for functionally significant heterodimerization (25)(26)(27). Furthermore, although there is an emerging theme in which dimerization contributes to high-affinity peptide binding and cAMP signaling (17,18), how dimerization contributes more globally to receptor signaling and whether it plays a role in ligand-directed stimulus bias are unknown.…”