Biology subjects

Bandekar, S. J.

Publications and source records attributed to Bandekar, S. J..

5 recordsLinked to original sources

bioRxiv2024

Structural analysis and conformational dynamics of a holo-adhesion GPCR reveal interplay between extracellular and transmembrane domains

Adhesion G Protein-Coupled Receptors (aGPCRs) are key cell-adhesion molecules involved in numerous physiological functions. aGPCRs have large multi-domain extracellular regions (ECR) containing a conserved GAIN domain that precedes their seven-pass transmembrane domain (7TM). Ligand binding and mechanical force applied on the ECR regulate receptor function. However, how the ECR communicates with the 7TM remains elusive, because the relative orientation and dynamics of the ECR and 7TM within a holoreceptor is unclear. Here, we describe the cryo-EM reconstruction of an aGPCR, Latrophilin3/ADGRL3, and reveal that the GAIN domain adopts a parallel orientation to the membrane and has constrained movement. Single-molecule FRET experiments unveil three slow-exchanging FRET states of the ECR relative to the 7TM within the holoreceptor. GAIN-targeted antibodies, and cancer-associated mutations at the GAIN-7TM interface, alter FRET states, cryo-EM conformations, and receptor signaling. Altogether, this data demonstrates conformational and functional coupling between the ECR and 7TM, suggesting an ECR-mediated mechanism of aGPCR activation.

biochemistry↗

bioRxiv2024

Molecular and computational studies of ADGRG6 reveal its extracellular CUB domain as a key regulator of cAMP signaling

Adhesion G protein-coupled receptors (aGPCRs) transduce extracellular adhesion events into cytoplasmic signaling pathways. ADGRG6/GPR126 is an aGPCR critical for axon myelination, heart development and ear development; ADGRG6 is also associated with developmental diseases and cancers. ADGRG6 has a large, alternatively spliced, five-domain extracellular region (ECR) that samples different conformations and is essential for receptor function in vivo. However, the mechanistic details of how the ECR regulates signaling are unclear. Herein, we studied the conformational dynamics of the conserved CUB domain which is located at the distal N-terminus of the ADGRG6 ECR and is deleted in an alternatively spliced isoform ({Delta}CUB). We show that the {Delta}CUB isoform has decreased signaling and is insensitive to inclusion of an activating splice insertion (+ss). Molecular dynamics simulations suggest that the CUB domain is involved in interdomain contacts to maintain a compact ECR conformation. A cancer-associated CUB domain mutant, C94Y, drastically perturbs the ECR conformation and results in elevated signaling, whereas another CUB mutant located near a conserved Ca2+-binding site, Y96A, decreases signaling. Our results suggest an ECR-mediated mechanism for ADGRG6 regulation in which the CUB domain instructs conformational changes within the ECR to regulate receptor signaling.

biochemistry↗

bioRxiv2024

Structure of the extracellular region of the adhesion GPCR CELSR1 reveals a compact module which regulates G protein-coupling

The Cadherin EGF Laminin G seven-pass G-type receptor subfamily (CELSR/ADGRC) is one of the most conserved among adhesion G protein-coupled receptors and is essential for animal development. The extracellular regions (ECRs) of CELSRs are large with 23 adhesion domains. However, molecular insight into CELSR function is sparsely available. Here, we report the 3.8 [A] cryo-EM reconstruction of the mouse CELSR1 ECR and reveal that 14 domains form a compact module mediated by conserved interactions majorly between the CADH9 and C-terminal GAIN domains. In the presence of Ca2+, the CELSR1 ECR forms a dimer species mediated by the cadherin repeats putatively in an antiparallel fashion. Cell-based assays reveal the N-terminal CADH1-8 repeat is required for cell-cell adhesion and the C-terminal CAHD9-GAIN compact module can regulate cellular adhesion. Our work provides molecular insight into how one of the largest GPCRs uses defined structural modules to regulate receptor function.

biochemistry↗

bioRxiv2023

The adhesion GPCRs CELSR1-3 and LPHN3 engage G proteins via distinct activation mechanisms

Adhesion GPCRs (aGPCRs) are a large GPCR class that direct diverse fundamental biological processes. One prominent mechanism for aGPCR agonism involves autoproteolytic cleavage, which generates an activating, membrane-proximal tethered agonist (TA). How universal this mechanism is for all aGPCRs is unclear. Here, we investigate G protein induction principles of aGPCRs using mammalian LPHN3 and CELSR1-3, members of two aGPCR families conserved from invertebrates to vertebrates. LPHNs and CELSRs mediate fundamental aspects of brain development, yet CELSR signaling mechanisms are unknown. We found that CELSR1 and CELSR3 are cleavage-deficient, while CELSR2 is efficiently cleaved. Despite differential autoproteolysis, CELSR1-3 all engage GS, and CELSR1 or CELSR3 TA point mutants retain GS coupling activity. CELSR2 autoproteolysis enhances GS coupling, yet acute TA exposure alone is insufficient. These studies support that aGPCRs signal via multiple paradigms and provide insights into CELSR biological function.

cell biology↗

bioRxiv2022

Isoform- and ligand-specific modulation of the adhesion GPCR ADGRL3/Latrophilin3 by a synthetic binder

Adhesion G protein-coupled receptors (aGPCRs) are cell-surface proteins with large extracellular regions that bind to multiple ligands to regulate key biological functions including neurodevelopment and organogenesis. Modulating a single function of a specific aGPCR isoform while affecting no other function and no other receptor is not trivial. Here, we engineered an antibody, termed LK30, that binds to the extracellular region of the aGPCR ADGRL3, and specifically acts as an agonist for ADGRL3 but not for its isoform, ADGRL1. The LK30/ADGRL3 complex structure revealed that the LK30 binding site on ADGRL3 overlaps with the binding site for an ADGRL3 ligand - teneurin. In cellular-adhesion assays, LK30 specifically broke the trans-cellular interaction of ADGRL3 with teneurin, but not with another ADGRL3 ligand - FLRT3. Our work provides proof of concept for the modulation of isoform- and ligand-specific aGPCR functions using unique tools, and thus establishes a foundation for the development of fine-tuned aGPCR-targeted therapeutics.

biochemistry↗