Research On The Cell Membrane Expression Pattern And Regulation Mechanism Of GABA_B Receptor In Drosophila Melanogaster

GABAB receptor is the metabotropic receptor of the important neurotransmitter ?-aminobutyric acid in the central nervous system.It is also the first identified heterodimeric G protein-coupled receptor family,which is consisted of GB1 and GB2 subunits.Each subunit is composed of an extracellular domain,a seven transmembrane domain and an intracellular domain.GABAB receptor is widely distributed on the cell membrane surface of pre-synapses and postsynapses,and perform cell signal function by coupling with intracellular heterotrimeric G protein G???.Scientific research has shown that mammalian GABAB receptors are related to various neurological and neuropsychiatric disorders,such as spasm,epilepsy,pain,addiction and anxiety,and are important targets for two clinical drugs.However,mammalian models have long individual life cycles,fuzzy boundaries between juvenile and adult morphology,high feeding costs,difficulty in large-scale experiments,and too many neurological determinants of individual behavior.It is difficult to accurately and in-depth study the molecular mechanisms of related pathological symptoms.Drosophila melanogaster(hereinafter referred to as Drosophila)is one of the commonly used model organisms in modern biological research.It has a low cost of breeding,a short life cycle,a clear boundary between larvae and adults,a nearly unlimited number of individuals can be obtained and it is easy to prapare large-scale genetic mutagenesis.So it has gradually been used in neuropharmacology research.Drosophila genome encodes three GABAB receptor subtypes,namely d GB1,d GB2 and d GB3 subunits.Among them,d GB1 and d GB2 are homologous genes of mammalian GB1 and GB2 subunits,respectively,however,no homologous genes of d GB3 have been found in mammals.Scientific research shows that Drosophila GABAB receptors are essential for regulating circadian rhythms and maintaining sleep,and can be used as models for certain neurobehavioral and neurological diseases such as alcohol addiction and pain.The assembly and cell surface expression of mammalian GABAB receptor heterodimer are strictly regulated,however,thses issues related to Drosophila GABAB receptor are rarely studies.It is not clear whether d GB1 or d GB2 alone can be expressed on the cell membrane surface alone.Focusing only on the GABAB receptor heterodimer and ignoring the cell membrane expression patterns and potential physiological functions of the d GB1 or d GB2 subunit alone is not sufficient to explain Drosophila GABAB receptor research model in detail.The present research focuses on the d GB1 and d GB2 subunits of the Drosophila GABAB receptor,and finds a completely opposite cell membrane expression pattern to mammals,that is,overexpression of d GB2 alone will be retained in the endoplasmic reticulum(ER),unless d GB2 and d GB1 are co-expressed to help d GB2 transport from the ER to the cell membrane surface together to form a functional GABAB receptor heterodimer.Further studies have found that the ER retention of the Drosophila d GB2 subunit is regulated by the second intracellular loop of its transmembrane domain,which is completely different from the ER retention of the mammalian GB1 subunit regulated by "Rs RR" and "EKSRLL" signal sequences.Next,the present research explores and determines that the specific interaction of the coiled-coil(hereinafter referred to as cc)domain of the C-terminal of d GB1 and d GB2 subunits directly mediates the assembly of heterodimers,which is similar to mammalian GABAB receptor.Lacking cc domain of any subunit fails to form heterodimeric GABAB receptor and to transport from ER to cell membrane surface.Furthermore,the present research uses modern structural biology techniques such as X-ray crystallography and nuclear magnetic resonance and determinates a homodimeric d GB2 cc domian crystal structure and the heterodimeric d GB1:d GB2 cc domain aqueous solution structure.Analyzing these structural models and comparing them with the previously reported mammalian GB1 and GB2 subunit cc crystal structure model,it is revealed that the interactions between these cc domain dimers are similar in general and mutually exclusive in details.In summary,the present research discovered a new cell membrane expression pattern and a new cell surface trafficking mode from the ER of the drosophila GABAB receptor heterodimer,and determinated structures of the elements that ragulate the hoterodimeric assembly and cell membrane targeting transports,confirming the conservation of the regulatory elements during evolution.The present research reveals that GABAB receptor keeps a quality control system that regulates functional heterodimer assembly and cell membrane expression during evolution,which will better promote the use of Drosophila models for GABAB receptor neuropharmacological research.