INTRODUCTION

Bilateral frontoparietal polymicrogyria (BFPP ) is an autosomal recessive genetic disorder, caused by the mutations in the GPR56/ADGRG1 gene located on human chromosome 16q21 [1, 2]. The genotype–phenotype analysis of BFPP patients yielded a cobblestone-like cortical malformation, which is characterized as formation of neuronal ectopias on the surface of the brain and aberrant migration of neural progenitor cells [3-5]. At the pial surface, the interaction of GPR56 with extracellular matrix protein collagen III activates the Rho signaling pathway through Gα12/13 and this results in inhibition of the neuronal migration [6].
GPR56 is a member of the G protein-coupled receptor (GPCR) superfamily. In humans, over 800 members constitute this huge receptor superfamily, hallmarked by their 7-transmembrane (7TM) structure and ability to couple heterotrimeric G proteins upon activation. GPCRs are involved in almost all biological processes with their pivotal role as cellular signal transduction of immensely diverse extracellular stimuli (i.e. photons, ions, small molecules, neurotransmitters, hormones and even mechanical stimuli) inside the cell. Mutations and functional problems in GPCRs are linked to many important human diseases [7]. Hence, taking part in almost all physiological processes and possessing druggable sites accessible at the cell surface have already made GPCRs targets of almost 40% of the prescribed drugs [8].
GPR56 is an adhesion GPCR (aGPCR) according to a further phylogenetic classification of GPCRs in the human genome [9, 10]. With 33 members, aGPCRs constitute the second largest GPCR family in humans. Almost all members in this receptor family have numerous splice variants and these receptor isoforms show tissue-dependent expression, various functions and different downstream signaling properties. All these features in aGPCRs adds another layer of complexity and structural variety to this enigmatic family of receptors [11-13]. Arguably, the most intriguing characteristics of aGPCRs are their extremely large N-terminus which consists of various protein domains that are involved in protein-protein, cell-cell and cell-matrix interactions and these multi-domain structures point to the multifaceted roles of this receptor family in signal transduction, modulation as well as cell adhesion [14, 15]. A very important characteristic feature of aGPCRs (except for GPR123/ADGRA1) is a highly conserved extracellular GPCR autoproteolysis-inducing (GAIN) domain [16]. In most aGPCRs, an autoproteolytic cleavage occurs at the GPCR proteolysis site (GPS) located within the GAIN domain, yielding non-covalently bound extracellular N-terminal fragment (NTF) and membrane integrated C-terminal fragment (CTF) composed of 7TM structure and theStachel peptide [17-22].
Activation of GPCRs on the extracellular side causes conformational changes that facilitate the interaction of receptors with G proteins or β-arrestins at the cytosolic side of the plasma membrane and mediate the downstream signaling pathways. Elucidating the molecular mechanisms of receptor activation and signaling with high spatiotemporal resolution is very crucial for GPCRs due to their pivotal roles in many physiological processes, their related pathologies, therapeutic relevance and druggability. During the past recent years, there has been a considerable effort to develop bioluminescent or fluorescent biosensors working on the principles of resonance energy transfer (RET) for the real-time monitoring of the GPCR activation in live cells [23-25]. These systems were successfully applied to study the activation of numerous GPCRs, their coupling with G proteins and recruitment of β-arrestins [26, 27].
In this work, the effects of BFPP mutations on GPR56 signaling upon Stachel peptide activation with Gα12, Gα13 and Gα11 were studied using BRET biosensors. In principle, biosensors used are based on the Gβγ dissociation from the G protein α-subunit upon GPCR activation in the exogenous expression of G protein-coupled receptor kinase (GRK) [28, 29]. β-arrestin recruitment BRET biosensor was employed to investigate the effects of disease-associated mutations on the arrestin recruitment for Stachel peptide activated receptors [30].