GPR114/ADGRG5 is activated by its tethered peptide agonist because it is a cleaved adhesion GPCR.

Family B2 or adhesion G protein-coupled receptors (AGPCRs) are distinguished by variable extracellular regions that contain a modular protease, termed the GPCR autoproteolysis-inducing domain that self-cleaves the receptor into an N-terminal fragment (NTF) and a C-terminal fragment (CTF), or seven transmembrane domain (7TM). The NTF and CTF remain bound after cleavage through noncovalent interactions. NTF binding to a ligand(s) presented by nearby cells, or the extracellular matrix anchors the NTF, such that cell movement generates force to induce NTF/CTF dissociation and expose the AGPCR tethered peptide agonist. The released tethered agonist (TA) binds rapidly to the 7TM orthosteric site to activate signaling. The orphan AGPCR, GPR114 was reported to be uncleaved, yet paradoxically capable of activation by its TA. GPR114 has an identical cleavage site and TA to efficiently cleave GPR56. Here, we used immunoblotting and biochemical assays to demonstrate that GPR114 is a cleaved receptor, and the self-cleavage is required for GPR114 TA-activation of Gs and no other classes of G proteins. Mutagenesis studies defined features of the GPR114 and GPR56 GAINA subdomains that influenced self-cleavage efficiency. Thrombin treatment of protease-activated receptor 1 leader/AGPCR fusion proteins demonstrated that acute decryption of the GPR114/56 TAs activated signaling. GPR114 was found to be expressed in an eosinophilic-like cancer cell line (EoL-1 cells) and endogenous GPR114 was efficiently self-cleaved. Application of GPR114 TA peptidomimetics to EoL-1 cells stimulated cAMP production. Our findings may aid future delineation of GPR114 function in eosinophil cAMP signaling related to migration, chemotaxis, or degranulation.

Mechanisms of adhesion G protein-coupled receptor activation.

Adhesion G protein-coupled receptors (AGPCRs) are a thirty-three-member subfamily of Class B GPCRs that control a wide array of physiological processes and are implicated in disease. AGPCRs uniquely contain large, self-proteolyzing extracellular regions that range from hundreds to thousands of residues in length. AGPCR autoproteolysis occurs within the extracellular GPCR autoproteolysis-inducing (GAIN) domain that is proximal to the N terminus of the G protein-coupling seven-transmembrane-spanning bundle. GAIN domain-mediated self-cleavage is constitutive and produces two-fragment holoreceptors that remain bound at the cell surface. It has been of recent interest to understand how AGPCRs are activated in relation to their two-fragment topologies. Dissociation of the AGPCR fragments stimulates G protein signaling through the action of the tethered-peptide agonist stalk that is occluded within the GAIN domain in the holoreceptor form. AGPCRs can also signal independently of fragment dissociation, and a few receptors possess GAIN domains incapable of self-proteolysis. This has resulted in complex theories as to how these receptors are activated in vivo, complicating pharmacological advances. Currently, there is no existing structure of an activated AGPCR to support any of the theories. Further confounding AGPCR research is that many of the receptors remain orphans and lack identified activating ligands. In this review, we provide a detailed layout of the current theorized modes of AGPCR activation with discussion of potential parallels to mechanisms used by other GPCR classes. We provide a classification means for the ligands that have been identified and discuss how these ligands may activate AGPCRs in physiological contexts.

Selective Covalent Targeting of Anti-apoptotic BFL-1 by a Sulfonium-Tethered Peptide.

Anti-apoptotic B cell lymphoma 2 (BCL-2) family proteins are proven targets for human cancers. Targeting the BH3-binding pockets of these anti-apoptotic proteins could reactivate apoptosis in BCL-2-depedent cancers. BFL-1 is a BCL-2 family protein overexpressed in various chemoresistant cancers. A unique cysteine at the binding interface of the BH3 and BFL-1 was previously proven to be an intriguing targeting site to irreversibly inhibit BFL-1 functions with stabilized cyclic peptide bearing a covalent warhead. Recently, we developed a sulfonium-tethered peptide cyclization strategy to construct peptide ligands that could selectively and efficiently react with the cysteine(s) of target proteins near the interacting interface. Using this method, we constructed a BFL-1 peptide inhibitor, B4-MC, that could selectively conjugate with BFL-1 both in vitro and in cell. B4-MC showed good cellular uptake, colocalized with BFL-1 on mitochondria, and showed obvious growth inhibition of BFL-1 over-expressed cancer cell lines.

Structural clarity is brought to adhesion G protein-coupled receptor tethered agonism.

An elusive problem in the adhesion G protein-coupled receptor (AGPCR) field is full understanding of the activation mechanisms of the 33-member receptor class. With the recent solution of active-state structures of nearly one quarter of AGPCRs, clarity has been brought to how AGPCRs are activated in response to endogenous full agonists. AGPCRs are self-activated via a tethered peptide agonist (TA) that transitions from a concealed or encrypted location to a decrypted state that binds to a typical GPCR orthosteric binding pocket. Here, we summarize the key milestones that led to the discovery of the AGPCR TA activation mechanism and discuss how extracellular shear forces may initiate TA decryption in physiological contexts. We compare the new active-state AGPCR structures and note that the orthosteric site-engaged TAs adopt a remarkably similar partial α-helical hook-like conformation, despite divergence of overall receptor similarity. Further, we contrast the TA-bound AGPCR structures to a partially active AGPCR structure to highlight the transitions AGPCRs may undergo during activation. Finally, we provide commentary on the validity of alternative AGPCR activation mechanisms.

The GPCR properties of polycystin-1- A new paradigm.

Polycystin-1 (PC1) is an 11-transmembrane (TM) domain-containing protein encoded by the PKD1 gene, the most frequently mutated gene leading to autosomal dominant polycystic kidney disease (ADPKD). This large (> 462 kDal) protein has a complex posttranslational maturation process, with over five proteolytic cleavages having been described, and is found at multiple cellular locations. The initial description of the binding and activation of heterotrimeric Gαi/o by the juxtamembrane region of the PC1 cytosolic C-terminal tail (C-tail) more than 20 years ago opened the door to investigations, and controversies, into PC1's potential function as a novel G protein-coupled receptor (GPCR). Subsequent biochemical and cellular-based assays supported an ability of the PC1 C-tail to bind numerous members of the Gα protein family and to either inhibit or activate G protein-dependent pathways involved in the regulation of ion channel activity, transcription factor activation, and apoptosis. More recent work has demonstrated an essential role for PC1-mediated G protein regulation in preventing kidney cyst development; however, the mechanisms by which PC1 regulates G protein activity continue to be discovered. Similarities between PC1 and the adhesion class of 7-TM GPCRs, most notably a conserved GPCR proteolysis site (GPS) before the first TM domain, which undergoes autocatalyzed proteolytic cleavage, suggest potential mechanisms for PC1-mediated regulation of G protein signaling. This article reviews the evidence supporting GPCR-like functions of PC1 and their relevance to cystic disease, discusses the involvement of GPS cleavage and potential ligands in regulating PC1 GPCR function, and explores potential connections between PC1 GPCR-like activity and regulation of the channel properties of the polycystin receptor-channel complex.

Opportunities and challenges for drug discovery in modulating Adhesion G protein-coupled receptor (GPCR) functions.

INTRODUCTION: The G protein-coupled receptors (GPCR) superfamily is among the most widely exploited targets for therapeutics, with drugs mainly targeting the Rhodopsin, Glutamate and Secretin family receptors. The receptors of the Adhesion family, however, remain comparatively unexplored in this aspect. This review aims to discuss the druggability of Adhesion GPCRs (aGPCR), highlighting the relevant opportunities and challenges. AREAS COVERED: In this review, the authors provide a disease-oriented summary of aGPCR involvement in humans and discuss the current status of characterizing therapeutic agents with a focus on new opportunities using low molecular weight substances. EXPERT OPINION: The small molecule antagonist dihydromunduletone and partial agonist 3-α-acetoxydihydrodeoxygedunin, along with the endogenous natural ligand synaptamide currently comprise some of the most important discoveries made in an attempt to characterize aGPCR druggability. The small molecule modulators provide important insights regarding the structure-activity relationship and suggest that targeting the tethered peptide agonist results in a nonselective pharmacological action, while synaptamide may be considered a potentially attractive tool to achieve a higher degree of selectivity.