Molecular sensing of mechano- and ligand-dependent adhesion GPCR dissociation.

Adhesion G-protein-coupled receptors (aGPCRs) bear notable similarity to Notch proteins1, a class of surface receptors poised for mechano-proteolytic activation2-4, including an evolutionarily conserved mechanism of cleavage5-8. However, so far there is no unifying explanation for why aGPCRs are autoproteolytically processed. Here we introduce a genetically encoded sensor system to detect the dissociation events of aGPCR heterodimers into their constituent N-terminal and C-terminal fragments (NTFs and CTFs, respectively). An NTF release sensor (NRS) of the neural latrophilin-type aGPCR Cirl (ADGRL)9-11, from Drosophila melanogaster, is stimulated by mechanical force. Cirl-NRS activation indicates that receptor dissociation occurs in neurons and cortex glial cells. The release of NTFs from cortex glial cells requires trans-interaction between Cirl and its ligand, the Toll-like receptor Tollo (Toll-8)12, on neural progenitor cells, whereas expressing Cirl and Tollo in cis suppresses dissociation of the aGPCR. This interaction is necessary to control the size of the neuroblast pool in the central nervous system. We conclude that receptor autoproteolysis enables non-cell-autonomous activities of aGPCRs, and that the dissociation of aGPCRs is controlled by their ligand expression profile and by mechanical force. The NRS system will be helpful in elucidating the physiological roles and signal modulators of aGPCRs, which constitute a large untapped reservoir of drug targets for cardiovascular, immune, neuropsychiatric and neoplastic diseases13.

7TM domain structures of adhesion GPCRs: what's new and what's missing?

Adhesion-type G protein-coupled receptors (aGPCRs) have long resisted approaches to resolve the structural details of their heptahelical transmembrane (7TM) domains. Single-particle cryogenic electron microscopy (cryo-EM) has recently produced aGPCR 7TM domain structures for ADGRD1, ADGRG1, ADGRG2, ADGRG3, ADGRG4, ADGRG5, ADGRF1, and ADGRL3. We review the unique properties, including the position and conformation of their activating tethered agonist (TA) and signaling motifs within the 7TM bundle, that the novel structures have helped to identify. We also discuss questions that the kaleidoscope of novel aGPCR 7TM domain structures have left unanswered. These concern the relative positions, orientations, and interactions of the 7TM and GPCR autoproteolysis-inducing (GAIN) domains with one another. Clarifying their interplay remains an important goal of future structural studies on aGPCRs.

Inhibition of adenylyl cyclase by GTPase-deficient Gαi is mechanistically different from that mediated by receptor-activated Gαi.

Signal transduction through G protein-coupled receptors (GPCRs) has been a major focus in cell biology for decades. Numerous disorders are associated with GPCRs that utilize Gi proteins to inhibit adenylyl cyclase (AC) as well as regulate other effectors. Several early studies have successfully defined the AC-interacting domains of several members of Gαi by measuring the loss of activity upon homologous replacements of putative regions of constitutive active Gαi mutants. However, whether such findings can indeed be translated into the context of a receptor-activated Gαi have not been rigorously verified. To address this issue, an array of known and new chimeric mutations was introduced into GTPase-deficient Q204L (QL) and R178C (RC) mutants of Gαi1, followed by examinations on their ability to inhibit AC. Surprisingly, most chimeras failed to abolish the constitutive activity brought on by the QL mutation, while some were able to eliminate the inhibitory activity of RC mutants. Receptor-mediated inhibition of AC was similarly observed in the same chimeric constructs harbouring the pertussis toxin (PTX)-resistant C351I mutation. Moreover, RC-bearing loss-of-function chimeras appeared to be hyper-deactivated by endogenous RGS protein. Molecular docking revealed a potential interaction between AC and the α3/ß5 loop of Gαi1. Subsequent cAMP assays support a cooperative action of the α3/ß5 loop, the α4 helix, and the α4/ß6 loop in mediating AC inhibition by Gαi1-i3. Our results unveiled a notable functional divergence between constitutively active mutants and receptor-activated Gαi1 to inhibit AC, and identified a previously unknown AC-interacting domain of Gαi subunits. These results collectively provide valuable insights on the mechanism of AC inhibition in the cellular environment.

Re-examining the 'Dissociation Model' of G protein activation from the perspective of Gßγ signaling.

G protein-coupled receptors (GPCRs) represent a major group of drug targets with tremendous pharmacological value. Signals arising from GPCRs are primarily transduced via two functional components of their corresponding G proteins, the Gα subunit and the Gßγ dimer that dissociate from each other upon activation of the heterotrimer (Gαßγ). The Gßγ dimer has become an increasingly popular subject in GPCR signaling, owing to its numerous effectors and notable roles in signal integration. Because Gßγ dimers participate in a wide range of intracellular processes that regulate cellular physiology, they are often implicated in the pathology of various diseases. Yet, one caveat to the current 'Dissociation Model' on GPCR signaling is that unequivocal Gßγ signals are biasedly detected with Gi/o -coupled receptors, while Gßγ signals from Gs - or Gq -coupled receptors seem to play an auxiliary role. In this review, we revisit the evidence for or against the 'Dissociation Model' and discuss in detail several hypotheses that may explain such disparity and provide alternative interpretations to accommodate the 'biased Gßγ signals' observed in different biological systems. The issue of whether unique combinations of Gßγ dimer can confer signaling specificity is also discussed in the context of physiological relevance.

Monoamine Biosynthesis via a Noncanonical Calcium-Activatable Aromatic Amino Acid Decarboxylase in Psilocybin Mushroom.

Aromatic l-amino acid decarboxylases (AAADs) are a phylogenetically diverse group of enzymes responsible for the decarboxylation of aromatic amino acid substrates into their corresponding aromatic arylalkylamines. AAADs have been extensively studied in mammals and plants as they catalyze the first step in the production of neurotransmitters and bioactive phytochemicals, respectively. Unlike mammals and plants, the hallucinogenic psilocybin mushroom Psilocybe cubensis reportedly employs an unrelated phosphatidylserine-decarboxylase-like enzyme to catalyze l-tryptophan decarboxylation, the first step in psilocybin biosynthesis. To explore the origin of this chemistry in psilocybin mushroom, we generated the first de novo transcriptomes of P. cubensis and investigated several putative l-tryptophan-decarboxylase-like enzymes. We report the biochemical characterization of a noncanonical AAAD from P. cubensis ( PcncAAAD) that exhibits substrate permissiveness toward l-phenylalanine, l-tyrosine, and l-tryptophan, as well as chloro-tryptophan derivatives. The crystal structure of PcncAAAD revealed the presence of a unique C-terminal appendage domain featuring a novel double-ß-barrel fold. This domain is required for PcncAAAD activity and regulates catalytic rate and thermal stability through calcium binding. PcncAAAD likely plays a role in psilocybin production in P. cubensis and offers a new tool for metabolic engineering of aromatic-amino-acid-derived natural products.