Intracellular G Protein-coupled Receptors Research Articles

The ocular albinism type 1 gene product, OA1, spans intracellular membranes 7 times

OA1 (GPR143) is a pigment cell-specific intracellular glycoprotein consisting of 404 amino acid residues that is mutated in patients with ocular albinism type 1, the most common form of ocular albinism. While its cellular localization is suggested to be endolysosomal and melanosomal, the physiological function of OA1 is currently unclear. Recent reports predicted that OA1 functions as a G protein coupled receptor (GPCR) based on its weak amino acid sequence similarity to known GPCRs, and on demonstration of GPCR activity in OA1 mislocalized to the plasma membrane. Because mislocalization of proteins is often caused by or induces defects in their proper folding/assembly, the significance of these studies remains unclear. A characteristic feature of GPCRs is a seven transmembrane domain structure. We analyzed the membrane topology of OA1 properly localized to intracellular lysosomal organelles in COS-1 cells. To accomplish this analysis, we established experimental conditions that allowed selective permeabilization of the plasma membrane while leaving endolysosomal membranes intact. Domains were mapped by the insertion of a hemagglutinin (HA) tag into the predicted cytosolic/luminal regions of OA1 molecule and the accessibility of tag to HA antibody was determined by immunofluorescence. HA-tagged lysosome associated membrane protein 1 (LAMP1), a type I membrane protein, was employed as a reporter for selective permeabilization of the plasma membrane. Our results show experimentally that the C-terminus of OA1 is directed to the cytoplasm and that the protein spans the intracellular membrane 7 times. Thus, OA1, properly localized intracellularly, is a 7 transmembrane domain integral membrane protein consistent with its putative role as an intracellular GPCR.

Intracrine signaling through lipid mediators and their cognate nuclear G-protein-coupled receptors: a paradigm based on PGE2, PAF, and LPA1receptorsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

Prostaglandins (PGs), platelet-activating factor (PAF), and lysophosphatidic acid (LPA) are ubiquitous lipid mediators that play important roles in inflammation, cardiovascular homeostasis, and immunity and are also known to modulate gene expression of specific pro-inflammatory genes. The mechanism of action of these lipids is thought to be primarily dependent on their specific plasma membrane receptors belonging to the superfamily of G-protein-coupled receptors (GPCR). Increasing evidence suggests the existence of a functional intracellular GPCR population. It has been proposed that immediate effects are mediated via cell surface receptors whereas long-term responses are dependent upon intracellular receptor effects. Indeed, receptors for PAF, LPA, and PGE2(specifically EP1, EP3, and EP4) localize at the cell nucleus of cerebral microvascular endothelial cells of newborn pigs, rat hepatocytes, and cells overexpressing each receptor. Stimulation of isolated nuclei with these lipids reveals biological functions including transcriptional regulation of major genes, namely c-fos, cylooxygenase-2, and endothelial as well as inducible nitric oxide synthase. In the present review, we shall focus on the nuclear localization and signaling of GPCRs recognizing PGE2, PAF, and LPA phospholipids as ligands. Mechanisms on how nuclear PGE2, PAF, and LPA receptors activate gene transcription and nuclear localization pathways are presented. Intracrine signaling for lipid mediators uncover novel pathways to elicit their effects; accordingly, intracellular GPCRs constitute a distinctive mode of action for gene regulation.

The ocular albinism type 1 (OA1) protein and the evidence for an intracellular signal transduction system involved in melanosome biogenesis

Ocular albinism type 1 is an X-linked disorder characterized by severe reduction of visual acuity, retinal hypopigmentation, foveal hypoplasia, optic misrouting and the presence of giant melanosomes (macromelanosomes) in skin melanocytes and retinal pigment epithelium. The protein product of the OA1 gene is a pigment cell specific membrane glycoprotein, displaying structural and functional features of G protein-coupled receptors (GPCRs). However, in contrast to all other previously characterized GPCRs, OA1 is not localized to the plasma membrane, but is targeted to intracellular organelles, namely late endosomes/lysosomes and melanosomes. These unique characteristics suggest that OA1 represents the first example described so far of an exclusively intracellular GPCR and regulates melanosome biogenesis by transducing signals from the organelle lumen to the cytosol. These findings support previous hypotheses that GPCR-mediated signaling might also operate at the internal membranes in mammalian cells.

Intracellular Signaling of Lipid Mediators via Cognate Nuclear G Protein–Coupled Receptors

Platelet-activating factor (PAF) and lysophosphatidic acid (LPA) are ubiquitous lipid mediators that play important roles in inflammation, cardiovascular homeostasis, and immunity and are also known to modulate gene expression of specific proinflammatory genes. The mechanism of action of these phospholipids is thought to be primarily dependent on their specific plasma membrane receptors belonging to the superfamily of G protein-coupled receptors (GPCRs). However, increasing evidence suggests the existence of a functional intracellular GPCR population. It has been suggested that immediate effects are mediated by cell surface receptors, whereas long-term responses are mediated by intracellular receptors. PAF and LPA(1) receptors localize at the cell nucleus of cerebral microvascular endothelial cells of newborn pig, rat hepatocytes, and cells overexpressing each receptor, and stimulation of isolated nuclei reveal biological functions, including transcriptional regulation of major genes, namely cylooxygenase-2 and inducible nitric oxide synthase. This mini review focuses on the nuclear localization and signaling of GPCRs, recognizing PAF and LPA phospholipids as ligands. Theories on how nuclear PAF and LPA1 receptors activate gene transcription and nuclear localization pathways are discussed. Intracrine signaling for lipid mediators uncover novel pathways to elicit their effects; moreover, intracellular GPCRs constitute a distinctive mode of action for gene regulation.

Ocular albinism: evidence for a defect in an intracellular signal transduction system.

G protein-coupled receptors (GPCRs) participate in the most common signal transduction system at the plasma membrane. The wide distribution of heterotrimeric G proteins in the internal membranes suggests that a similar signalling mechanism might also be used at intracellular locations. We provide here structural evidence that the protein product of the ocular albinism type 1 gene (OA1), a pigment cell-specific integral membrane glycoprotein, represents a novel member of the GPCR superfamily and demonstrate that it binds heterotrimeric G proteins. Moreover, we show that OA1 is not found at the plasma membrane, being instead targeted to specialized intracellular organelles, the melanosomes. Our data suggest that OA1 represents the first example of an exclusively intracellular GPCR and support the hypothesis that GPCR-mediated signal transduction systems also operate at the internal membranes in mammalian cells.