The adenosine A1 receptor (A1R) is a G protein-coupled receptor (GPCR) for adenosine, a ubiquitous neuromodulator, and thus regulates neuronal excitability, as well as arousal and sensitivity to pain. In addition, we have previously described a new mode of action for A1R: in cerebellar Purkinje cells, its activation attenuates neuronal responses to glutamate, as mediated by the type-1 metabotropic glutamate receptor (mGluR1). mGluR1 is also a GPCR, and elicits such responses as long-term depression of the postsynaptic response to glutamate, a cellular basis for cerebellar motor learning. Here, we explore in greater detail the interaction between A1R and mGluR1 using non-neuronal cells. Co-immunoprecipitation and Förster resonance energy transfer (FRET) analysis reveal that A1R and mGluR1 form a complex. Furthermore, we found that mGluR1 activation inhibits A1R signaling, as measured by changes in intracellular cAMP. These findings demonstrate that A1R and mGluR1 have the intrinsic ability to form a heteromeric complex and mutually modulate signaling. This interaction may represent a new form of intriguing GPCR-mediated cellular responses.
Background: Mechanical alloknesis (or innocuous mechanical stimuli-evoked itch) often occurs in dry skin-based disorders such as atopic dermatitis and psoriasis. However, the molecular and cellular mechanisms underlying mechanical alloknesis remain unclear. We recently reported the involvement of CD26 in the regulation of psoriatic itch. This molecule exhibits dipeptidyl peptidase IV (DPPIV) enzyme activity and exerts its biologic effects by processing various substances, including neuropeptides. Objective: The aim of the present study was to investigate the peripheral mechanisms of mechanical alloknesis by using CD26/ DPPIV knockout (CD26KO) mice. Methods: We applied innocuous mechanical stimuli to CD26KO or wild-type mice. The total number of scratching responses was counted as the alloknesis score. Immunohistochemical and behavioral pharmacologic analyses were then performed to examine the physiologic activities of CD26/DPPIV or endomorphins (EMs), endogenous agonists of m-opioid receptors. Results: Mechanical alloknesis was more frequent in CD26KO mice than in wild-type mice. The alloknesis score in CD26KO mice was significantly reduced by the intradermal administration of recombinant DPPIV or naloxone methiodide, a peripheral m-opioid receptor antagonist, but not by that of mutant DPPIV without enzyme activity. EMs (EM-1 and EM-2), selective ligands for m-opioid receptors, are substrates for DPPIV. Immunohistochemically, EMs were located in keratinocytes, fibroblasts, and peripheral sensory nerves. Behavioral analyses revealed that EMs preferentially provoked mechanical alloknesis over chemical itch. DPPIV-digested forms of EMs did not induce mechanical alloknesis. Conclusion: The present results suggest that EMs induce mechanical alloknesis at the periphery under the enzymatic control of CD26/DPPIV. (J Allergy Clin Immunol 2021;nnn:nnn-nnn.)
Molecular networks containing various proteins mediate many types of cellular processes. Elucidation of how the proteins interact will improve our understanding of the molecular integration and physiological and pharmacological propensities of the network. One of the most complicated and unexplained interactions between proteins is the inter-G protein-coupled receptor (GPCR) interaction. Recently, many studies have suggested that an interaction between neurotransmitter GPCRs may mediate diverse modalities of neural responses. The B-type gamma-aminobutyric acid (GABA) receptor (GBR) and type-1 metabotropic glutamate receptor (mGluR1) are GPCRs for GABA and glutamate, respectively, and each plays distinct roles in controlling neurotransmission. We have previously reported the possibility of their functional interaction in central neurons. Here, we examined the interaction of these GPCRs using stable cell lines and rat cerebella. Cell-surface imaging and coimmunoprecipitation analysis revealed that these GPCRs interact on the cell surface. Furthermore, fluorometry revealed that these GPCRs mutually modulate signal transduction. These findings provide solid evidence that mGluR1 and GBR have intrinsic abilities to form complexes and to mutually modulate signaling. These findings indicate that synaptic plasticity relies on a network of proteins far more complex than previously assumed.
G-protein-coupled receptors (GPCRs) may form homomeric or heteromeric complexes and cooperatively mediate intracellular responses. Previously, we showed modulation of type 1 metabotropic glutamate receptor (mGluR1) function by metabotropic gamma-aminobutyric acid receptor (GABA B R) in cerebellar Purkinje cells. The activity of mGluR1 is mediated by a G q protein, and has a crucial role in synaptic plasticity and motor learning. GABA B R inhibits neuronal activity through G i protein, which regulates the release of neurotransmitters and the activity of ion channels. In this report, we investigated in greater detail the relationship of these GPCRs using non-neuronal cells. Using live cell imaging and biochemical analysis, we showed that mGluR1 and GABA B R form complexes at the cell surface. Moreover, using cAMP homogenous luminescence assay and calcium imaging, we found that mGluR1 and GABA B R regulate their signal transduction of each other. These findings provide a new insight into neuronal GPCR signaling and demonstrate a novel regulatory mechanism of synaptic transmission. This interaction would be involved in several important physiological and pathophysiological functions related to mGluR1 and GABA B R, such as cerebellar motor learning and its dysfunction.
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