Mutual regulation between metabotropic glutamate type 1α receptor and caveolin proteins: from traffick to constitutive activity
Introduction
Glutamate is the major excitatory neurotransmitter in the mammalian brain involved in learning and memory as well as in neurotoxicity, playing a critical role in the development or progression of diverse neurological disorders. This amino acid acts at multiple receptor types divided into two main groups, namely the ionotropic glutamate receptors, which form ion channels and mediate fast excitatory glutamate responses, and metabotropic glutamate (mGlu) receptors, that mediate slower glutamate responses by coupling to the intracellular signal transduction pathways via G proteins [1]. So far, eight members of the mGlu receptor family have been identified and categorized into three subgroups on the basis of their sequence homology, agonist selectivity and signal transduction pathway. Group I contains mGlu1 and mGlu5 receptors subtypes, which are coupled to phospholipase C in transfected cells, and have quisqualic acid (Quis) as their most potent agonist. The second group consists of mGlu2 and mGlu3 receptors, which couple negatively to adenylyl cyclase in transfected cells and for which l-2-(carboxycyclopropyl) glycine is the most potent agonist. Group III contains mGlu4, mGlu6, mGlu7 and mGlu8 receptors, which again couple negatively to adenylyl cyclase and have l-2-amino-4-phosphonobutyric acid as their most potent agonist. In the last decade, it was shown that group I mGlu receptors exhibit differential constitutive activity when expressed in heterologous systems as a change in the equilibrium between the inactive and active conformation of the receptor [2]. More recently, the presence of Homer proteins, which bind directly to the C-terminal tail of group I mGlu receptors, has been reported to be of high importance but not essential to control this agonist-independent activity in cerebellar cultured neurons and HEK-293 cells [3].
Caveolin proteins form the structural network of caveolae, which are small membrane microdomains of the plasma membrane thought to play a critical role in functions such as signal transduction, cholesterol transport, and endocytosis [4], [5], [6], [7]. It has been reported the presence of at least three mammalian subtypes of caveolins which show similarities in structure and function but with differential tissue distribution. Caveolin-1 and caveolin-2 show a similar tissue distribution whereas caveolin-3 is specifically distributed in the muscle tissue and may replace caveolin-1 as the major caveolar protein in differentiated muscle cells [8], [9]. While caveolin-1 and caveolin-3 can reach the plasma membrane, caveolin-2 is retained in the Golgi apparatus. Caveolin-1 expression rescues caveolin-2 and delivers it to the cell surface [10], [11], [12]. Moreover, several lines of evidence suggest that caveolin-1 controls a variety of plasma membrane-initiated signaling cascades acting as a direct regulator of the proteins that can be segregated into caveolae [5]. On the other hand, a shorter isoform of caveolin-2, caveolin-2β, is found surrounding intracellular lipid accumulations namely lipid droplets or lipid bodies and although its function has not yet been described, a role in lipid trafficking has been suggested [13].
In a previous report, we presented evidence of the presence of mGlu1α receptor within low-density caveolin-enriched plasma membrane fractions, being the receptor able to interact with caveolin-1 and caveolin-2 [14]. In the present study, a mutual functional interaction between mGlu1α receptor and caveolins is described. The expression of mGlu1α receptor recruits caveolin-2β and redirects it from intracellular lipid droplets to the plasma membrane and, on the other hand, the interaction of mGlu1α with caveolin-1 suppresses the constitutive activity of mGlu1α receptor.
Section snippets
Antibodies
The primary antibodies were anti-β-tubulin monoclonal antibody (Clone TUB 2.1, Sigma), affinity purified anti-Flag monoclonal antibody (Clone M2, Sigma), affinity purified anti-mGlu1 receptor polyclonal antibody F1-Ab (pan-mGluR1) [15], affinity purified anti-mGlu1α receptor polyclonal antibody F2-Ab [16], anti-caveolin-1 and anti-caveolin-2 antibodies (Transduction Laboratories) and mouse antitransferrin receptor antibody (Clone H68.4, Zymed). The secondary antibodies were
mGlu1α receptor associates with caveolin-enriched membrane fractions
In BHK-1α cells permanently transfected with rat mGlu1α receptor, an overlapping distribution between mGlu1α receptor and caveolins has been previously found [14]. By double immunolabeling experiments in transiently transfected HEK-293 cells, the subcellular distribution of mGlu1α receptor, caveolin-1, caveolin-2α and caveolin-2β was studied. As observed in Fig. 1, the overlap of the images from cells cotransfected with mGlu1α receptor and caveolin-1 resulted in a high degree of colocalization
Discussion
The results presented here show that mGlu1α receptor colocalizes, cofractionates in a low-density fraction and coimmunoprecipitates with caveolin-1 and caveolin-2β in transiently transfected HEK-293 cells. The interaction of mGlu1α receptor with caveolin-2β is able to modify the later subcellular distribution and the interaction between mGlu1α receptor with caveolin-1 affects the receptor constitutive activity.
In this study, the subcellular distribution of caveolin-2β in transfected HEK-293
Acknowledgments
F. Ciruela is currently holding a Ramón y Cajal research contract signed with the Ministerio de Ciencia y Tecnología. This study was supported by grants from Ministerio de Ciencia y Tecnología (BIO99-0601-C02-02, SAF2002-03293 to R.F. and SAF2001-3474 to E.C.), Fundació La Marató de TV3 (Marató/2001/012710 to R.F.) and European Community (QLRT-2000-01056 to R.F.). We are grateful to the personnel from Serveis Científic i Tècnics de la Universitat de Barcelona for their excellent technical
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These two authors have contributed equally to this work.