Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling

Abstract

Although the importance of the C terminus of the ␣ subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the G␣ subunit (G␣ peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between G␣ peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate G␣ peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate G␣ peptide stabilizes the agonistbound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (G␣ s /G␣ q -Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the ␤2-adrenergic receptor (␤2-AR)-G␣ s and V 1A receptor (V 1A R)-G␣ q . The G␣ s and G␣ q peptides adopt different orientations in ␤2-AR and V 1A R, respectively. The ␤2-AR/G␣ s peptide interface is dominated by electrostatic interactions, whereas the V 1A R/G␣ q peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in G␣ q prevents this peptide from interacting strongly with ␤2-AR. Mutagenesis to the G␣ s counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions.In recent years, there has been significant progress in structural and spectroscopic studies of Class A G protein-coupled receptors (GPCR), 3 which are energizing structure-based drug discovery efforts (1-8). Although these studies clearly demonstrate ligand-dependent structural changes in the GPCR, there remains a paucity of information on how GPCR conformation translates to selective G protein activation (9). Without structural information to compare and contrast multiple GPCR/G protein interfaces, the underlying mechanisms of selection remain incompletely understood. Currently, there is only one crystal structure of the complete GPCR/G protein interface (␤2-AR⅐G s complex) that provides a single essential snapshot of a highly dynamic interaction (10). Hence, alternative approaches are essential to dissect the structural elements within both the GPCR and its effectors that confer signaling specificity within the cellular environment. In this study, we combine FRETbased measurements of the GPCR/G protein interface in live cells, combined with computational modeling to build a rational scalable approach to identify structural hot spots that drive effector selection.One critical and well characterized component of the GPCR/G prote...

Receptor selectivity between the G proteins Gα 12 and Gα 13 is defined by a single leucine‐to‐isoleucine variation

“…). Based on similar Fluorescence Resonance Energy Transfer‐based SPASM sensors for other GPCRs (28, 40) and structural insights into the mechanisms of interactions between GPCRs and G proteins a subunits (7–12) we anticipated that following construct expression agonist‐induced engagement of the G protein segment with the receptor would result in enhanced BRET signal. We cloned these sensors into Flp‐In T‐REx 293 cells and, following doxycycline‐induced expression, studied initially the kinetics of response of the Gα 13 ‐containing sensor upon addition of zaprinast.…”

“…Certain GPCRs do, however, appear to display considerable selectivity for G 12 /G 13 over other G proteins, and in the case of the nominally orphan receptor GPR35 it has been suggested that there is even marked selectivity for G 13 over G 12 (49). To assess this in a controlled and potentially quantitative manner, we established a range of SPASM sensors (28, 40) in which hGPR35a was linked to the C‐terminal 27 aa of various G protein α subunits via a flexible linker in which receptor interaction with the G protein segment results in enhanced BRET signal. A variant NP form, lacking the G protein segment, did not result in altered BRET with addition of various agonists at hGPR35, demonstrating that the G protein segment was indeed required for signal alteration.…”

“…To assess this in a controlled and potentially quantitative manner, we established a range of SPASM sensors (28,40) in which hGPR35a was linked to the C-terminal 27 aa of various G protein a subunits via a flexible linker in which receptor interaction with the G protein segment results in enhanced BRET signal. A variant NP form, lacking the G protein segment, did not result in altered BRET with addition of various agonists at hGPR35, demonstrating that the G protein segment was indeed required for signal alteration.…”

“…Kleinau et al (51) have taken a mutagenic approach to address selective interactions of the thyrotropin receptor with G s and with G q , but these studies focused on the receptor rather than the G protein. A study with a larger direct relevance used the SPASM approach also employed herein to assess differences in interactions between receptors with Ga s and Ga q , 2 still markedly different G proteins from different families (40). Furthermore, an extensive computational analysis on the potential basis for GPCR-G protein selectivity by Flock et al (36) explored a potential barcode for receptor selectivity within the amino acid sequences at the C-terminal region of different G proteins.…”

Conformational plasticity of the intracellular cavity of GPCR−G-protein complexes leads to G-protein promiscuity and selectivity

“…Seminal works have shed light on the critical involvement of GPCR intracellular (IC) loops and the transmembrane (TM) helix 6 (TM6) interface in mediating selective G-protein interactions (19)(20)(21)(22)(23). Analysis of 3D structures combined with previous cell-based assay studies show the α5 helix in the C terminus of the Gα protein exhibits a large effect on selective coupling to GPCRs (24)(25)(26)(27)(28)(29). Here we study the dynamic interactions of the C terminus of Gα s , Gα i , and Gα q proteins, hereafter referred to as s-pep, i-pep, and q-pep, in combination with seven class A GPCRs (β2-Adrenergic Receptor, β 2 AR; β3-Adrenergic Receptor, β 3 AR; Dopamine 1 Receptor, D 1 R; α2A-Adrenergic Receptor, α 2A AR; Cannabinoid 1 Receptor, CB 1 R; α1A-Adrenergic Receptor, α 1A AR; and Vasopressin 1A Receptor, V 1A R), to delineate the GPCR−G-protein selectivity determinants.…”