Constitutive Formation of Oligomeric Complexes between Family B G Protein-Coupled Vasoactive Intestinal Polypeptide and Secretin Receptors

Abstract

Formation of oligomeric complexes of family A G protein-coupled receptors has been shown to influence their function and regulation. However, little is known about the existence of such complexes for family B receptors in this superfamily. We previously used bioluminescence resonance energy transfer (BRET) to demonstrate that the prototypic family B secretin receptor forms ligand-independent oligomeric complexes. Here, we show that subtypes of human vasoactive intestinal polypeptide receptors (VPAC1 and VPAC2) that represent the closest structurally related receptors to the secretin receptor also form constitutive oligomers with themselves and with the secretin receptor. We prepared tagged constructs expressing Renilla reniformis luciferase, yellow fluorescent protein, or cyan fluorescent protein at the carboxyl terminus of VPAC1, VPAC2, and secretin receptors, and performed BRET and morphologic fluorescence resonance energy transfer (FRET) studies with all combinations. The specificity of the BRET and FRET signals was confirmed by control studies. These constructs bound their natural ligands specifically and saturably, with these agonists able to elicit full cAMP responses. BRET studies showed that, like the secretin receptor, both VPAC receptors exhibited constitutive homo-oligomerization in COS cells. Unlike secretin receptor oligomers that were unaffected by ligand binding, the VPAC receptor homo-oligomers were modulated by vasoactive intestinal polypeptide. In addition, each of these three receptors formed hetero-oligomers with each other. The VPAC1-VPAC2 hetero-oligomers were modulated by vasoactive intestinal polypeptide binding, whereas the secretin-VPAC1 and secretin-VPAC2 receptor hetero-oligomers were unaffected by ligand treatment. Morphologic FRET studies demonstrated that each of the homo-oligomers and the VPAC1-VPAC2 receptor hetero-oligomers reached the cell surface, where receptor interactions were clear. However, coexpression of secretin receptors with either type of VPAC receptor resulted in intracellular trapping of the hetero-oligomeric complexes within the biosynthetic pathway. These studies provide new insight into the ability of family B G protein-coupled receptors to associate with each other in cells.Plasma membrane receptors represent a critically important interface between circulating and extracellular hormones and neurotransmitters and the cell's intracellular signaling cascades and effector machinery. We are beginning to recognize that the specificity and activity of ligand-receptor interaction at this critical interface can be affected by a variety of factors, including covalent modifications of the receptor and bimolecular interactions with it. One group of such interactions is the dimerization or oligomerization with the same or other receptors

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

“…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.…”

“…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.…”

Secretin Receptor Oligomers Form Intracellularly during Maturation through Receptor Core Domains

“…The same mutations also were incorporated into Rlu and YFP carboxyl-terminally tagged versions of the same SecR coding sequence, minus the 5' and 3' untranslated regions (35). Construction of pcDNA3/SecR-CFP (27) and pcDNA3/CCKBR-YFP (36) were described previously.…”

“…After three washes in PBS, the cells were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and then observed and photographed with the FRET epifluorescence microscope setup described in (27). Images for the dedicated CFP, YFP, and FRET filter channels were acquired at equivalent exposure times and gain settings that were adjusted for each sample to provide a maximum range of intensity values with a minimum amount of pixel saturation.…”