RESUMEN
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.
Asunto(s)
Receptores Acoplados a Proteínas G , Transducción de Señal , Adhesión Celular , Péptidos/farmacología , Péptidos/metabolismo , Unión Proteica , Dominios Proteicos , Receptores Acoplados a Proteínas G/química , HumanosRESUMEN
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.
Asunto(s)
Péptidos , Receptores Acoplados a Proteínas G , Adhesión Celular , Relación Estructura-Actividad , Receptores Acoplados a Proteínas G/metabolismo , Dominios y Motivos de Interacción de ProteínasRESUMEN
AIMS: Cardiac contractility, essential to maintaining proper cardiac output and circulation, is regulated by G protein-coupled receptor (GPCR) signaling. Previously, the absence of regulator of G protein signaling (RGS) 2 and 5, separately, was shown to cause G protein dysregulation, contributing to modest blood pressure elevation and exaggerated cardiac hypertrophic response to pressure-overload. Whether RGS2 and 5 redundantly control G protein signaling to maintain cardiovascular homeostasis is unknown. Here we examined how the dual absence of RGS2 and 5 (Rgs2/5 dbKO) affects blood pressure and cardiac structure and function. METHODS AND RESULTS: We found that Rgs2/5 dbKO mice showed left ventricular dilatation at baseline by echocardiography. Cardiac contractile response to dobutamine stress test was sex-dependently reduced in male Rgs2/5 dbKO relative to WT mice. When subjected to surgery-induced stress, male Rgs2/5 dbKO mice had 75% mortality within 72-96â¯h after surgery, accompanied by elevated baseline blood pressure and decreased cardiac contractile function. At the cellular level, cardiomyocytes (CM) from Rgs2/5 dbKO mice showed augmented Ca2+ transients and increased incidence of arrhythmia without augmented contractile response to electrical field stimulation (EFS) and activation of ß-adrenergic receptors (ßAR) with isoproterenol. Dual loss of Rgs2 and 5 suppressed forskolin-induced cAMP production, which was restored by Gi/o inactivation with pertussis toxin that also reduced arrhythmogenesis during EFS or ßAR stimulation. Cardiomyocyte NCX and PMCA mRNA expression was unaffected in Rgs2/5 dbKO male mice. However, there was an exaggerated elevation of EFS-induced cytoplasmic Ca2+ in the presence of SERCA blockade with thapsigargin. CONCLUSIONS: We conclude that RGS2 and 5 promote normal ventricular rhythm by coordinating their regulatory activity towards Gi/o signaling and facilitating cardiomyocyte calcium handling.