Highlights d GRKs and arrestins mediate the fast and slow desensitization of a subset of mGluRs d TIRF imaging reveals scaffold and catalytic coupling between mGluR3 and b-arrestins d Different patterns of Ser and Thr control the desensitization of group II mGluRs d Mutational analysis reveals G protein-and b-arrestin-biased mGluR3 variants

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Kir3 (or GIRK) channels have been known for nearly three decades to be activated by direct interactions with the βγ subunits of heterotrimeric G (Gαβγ) proteins in a membrane-delimited manner. Gα also interacts with GIRK channels and since PTX-sensitive Gα subunits show higher affinity of interaction they confer signaling specificity to G Protein-Coupled Receptors (GPCRs) that normally couple to these G protein subunits. In heterologous systems, overexpression of non PTX-sensitive Gα subunits scavenges the available Gβγ and biases GIRK activation through GPCRs that couple to these Gα subunits. Moreover, all Kir channels rely on their direct interactions with the phospholipid PIP2 to maintain their activity. Thus, signals that activate phospholipase C (e.g. through Gq signaling) to hydrolyze PIP2 result in inhibition of Kir channel activity. In this review, we illustrate with experiments performed in Xenopus oocytes that Kir channels can be used efficiently as reporters of GPCR function through Gi, Gs or Gq signaling. The membrane-delimited nature of this expression system makes it highly efficient for constructing dose-response curves yielding highly reproducible apparent affinities of different ligands for each GPCR tested.

The metabotropic glutamate receptors (mGluRs) form a family of neuromodulatory G protein-coupled receptors that contain both a seven-helix transmembrane domain (TMD) and a large extracellular ligand-binding domain (LBD) which enables stable dimerization. While numerous studies have revealed variability across subtypes in the initial activation steps at the level of LBD dimers, an understanding of inter-TMD interaction and rearrangement remains limited. Here we use a combination of single molecule fluorescence, molecular dynamics, functional assays, and conformational sensors to reveal that distinct TMD assembly properties drive differences between mGluR subtypes. We uncover a variable region within transmembrane helix 4 (TM4) that contributes to homo- and heterodimerization in a subtype-specific manner and tunes orthosteric, allosteric and basal activation. We also confirm a critical role for a conserved inter-TM6 interface in stabilizing the active state during orthosteric or allosteric activation. Together this study shows that inter-TMD assembly and dynamic rearrangement drive mGluR function with distinct properties between subtypes.

The metabotropic glutamate receptors (mGluRs) form a family of neuromodulatory G protein-coupled receptors that contain both a seven-helix transmembrane domain (TMD) and a large extracellular ligand-binding domain (LBD) which enables stable dimerization. While numerous studies have revealed variability across subtypes in the initial activation steps at the level of LBD dimers, an understanding of inter-TMD interaction and rearrangement remains limited. Here we use a combination of single molecule fluorescence, molecular dynamics, functional assays, and conformational sensors to reveal that distinct TMD assembly properties drive differences between mGluR subtypes. We uncover a variable region within transmembrane helix 4 (TM4) that contributes to homo- and heterodimerization in a subtype-specific manner and tunes orthosteric, allosteric and basal activation. We also confirm a critical role for a conserved inter-TM6 interface in stabilizing the active state during orthosteric or allosteric activation. Together this study informs a working model of inter-TMD rearrangement that drives mGluR function.

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Numerous processes contribute to the regulation of G protein–coupled receptors (GPCRs), but relatively little is known about rapid mechanisms that control signaling on the seconds time scale or regulate cross-talk between receptors. Here, we reveal that the ability of some GPCR kinases (GRKs) to bind Gα q both drives acute signaling desensitization and regulates functional interactions between GPCRs. GRK2/3-mediated acute desensitization occurs within seconds, is rapidly reversible, and can occur upon local, subcellular activation. This rapid desensitization is kinase independent, insensitive to pharmacological inhibition, and generalizable across receptor families and effectors. We also find that the ability of GRK2 to bind G proteins also enables it to regulate the extent and timing of Gα q -dependent signaling cross-talk between GPCRs. Last, we find that G protein/GRK2 interactions enable a novel form of GPCR trafficking cross-talk. Together, this work reveals potent forms of Gα q -dependent GPCR regulation with wide-ranging pharmacological and physiological implications.