INTRODUCTION
Transmembrane signaling of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) is mediated by ligand-dependent conformational changes that are transmitted from the extracellular ligand binding site to the intracellular side of the receptor to allow coupling with transducers. One hallmark of GPCR activation is the outward movement of the cytoplasmic end of transmembrane domain 6 (TM6) that opens up an intracellular cavity to accommodate the Gα subunit, leading to nucleotide exchange and activation of the G protein. Comparison of family A and family B receptor-Gs protein complex structures has revealed substantial differences in the conformational changes of TM6 upon activation. In family B GPCRs, TM6 shows a disruption of the helical fold and the formation of a sharp kink. This differs from the gradual bending in TM6 observed in family A GPCRs.
RATIONALE
Despite the recent surge of determined GPCR–G protein complex structures, the activation mechanism of family B receptors remains poorly understood. The missing conserved structural motifs found in family A GPCRs together with the marked differences in the conformation of TM6 in the active state suggest distinct activation mechanisms between family B and family A GPCRs. In particular, the disruption of the helical fold and the unraveling of the extracellular end of TM6 suggest that the energy required to produce the fully active-state of family B GPCRs is higher than for family A GPCRs. We investigated the functional impact of these structural differences by comparing the structure and function of a prototypical family B receptor, the glucagon receptor (GCGR), with the β2 adrenergic receptor (β2AR), a family A GPCR.
RESULTS
We present the cryo–electron microscopy structure of the GCGR-Gs complex bound to an engineered soluble glucagon derivative. The structure shows that full activation of GCGR leads to a disruption in the α-helix of TM6 typical for family B GPCRs. Analysis of the functional consequence of this helix break on receptor-mediated G protein dissociation and guanosine triphosphate (GTP) turnover reveals that GCGR exhibits a substantially lower guanine nucleotide exchange factor (GEF) activity in comparison with the family A receptor β2AR. Characterization of G protein association, guanosine diphosphate (GDP) release, and GTP binding kinetics shows that the receptor-mediated GDP dissociation and GTP binding of Gs are slower for GCGR than for β2AR. Measurements of ligand-dependent conformational alterations of GCGR by means of fluorescence and double electron-electron resonance spectroscopy show that agonist binding alone is insufficient to promote TM6 opening, in contrast to previously studied family A GPCRs, including β2AR. The outward movement of TM6 of GCGR is only observed upon interaction with Gs, suggesting that TM6 activation is only triggered by the engagement of the α5 helix of Gαs. Furthermore, TM6 of GCGR remains in the active state for a prolonged time after disengagement of Gs, which might contribute to the persistent and sustained cyclic adenosine monophosphate (cAMP) signaling previously observed for this receptor. A comprehensive comparison of the G protein activation kinetics for a number of other family A and family B GPCRs shows that family B receptors are in general less efficient than family A GPCRs in triggering G protein signaling.
CONCLUSION
Our findings provide evidence for distinct activation mechanisms between family A and family B GPCRs. We propose that formation of the helix break and the sharp kink in TM6 of GCGR requires overcoming a higher energy barrier than the bending and outward movement of TM6 in family A receptors. Because of this kinetic barrier, ligand binding alone is not sufficient to stabilize the outward movement of TM6 but promotes the initial formation of the receptor–G protein complex and subsequent full engagement of the G protein at a slower time scale. Once activated by the insertion of the α5 helix of Gαs into the receptor core, as seen in the nucleotide-free complex structure, TM6 stays in the active conformation long after full disengagement of the G protein. This may be responsible for the previously described sustained and prolonged signaling of GCGR. Our comprehensive comparison of the G protein activation kinetics of family A and family B receptors suggests that the activation mechanism described for GCGR is generalizable to other family B GPCRs.