The decrease in plasticity that occurs in the central nervous system during postnatal development is accompanied by the appearance of perineuronal nets (PNNs) around the cell body and dendrites of many classes of neuron. These structures are composed of extracellular matrix molecules, such as chondroitin sulfate proteoglycans (CSPGs), hyaluronan (HA), tenascin-R, and link proteins. To elucidate the role played by neurons and glial cells in constructing PNNs, we studied the expression of PNN components in the adult rat cerebellum by immunohistochemistry and in situ hybridization. In the deep cerebellar nuclei, only large excitatory neurons were surrounded by nets, which contained the CSPGs aggrecan, neurocan, brevican, versican, and phosphacan, along with tenascin-R and HA. Whereas both net-bearing neurons and glial cells were the sources of CSPGs and tenascin-R, only the neurons expressed the mRNA for HA synthases (HASs), cartilage link protein, and link protein Bral2. In the cerebellar cortex, Golgi neurons possessed PNNs and also synthesized HASs, cartilage link protein, and Bral2 mRNAs. To see whether HA might link PNNs to the neuronal cell surface by binding to a receptor, we investigated the expression of the HA receptors CD44, RHAMM, and LYVE-1. No immunolabelling for HA receptors on the membrane of net-bearing neurons was found. We therefore propose that HASs, which can retain HA on the cell surface, may act as a link between PNNs and neurons. Thus, HAS and link proteins might be key molecules for PNN formation and stability.

It is becoming clear that receptors that initiate signal transduction by interacting with G-proteins do not function as monomers, but often require accessory proteins for function. Some of these accessory proteins are chaperones, required for correct transport of the receptor to the cell surface, but the function of many accessory proteins remains unknown. We determined the role of an accessory protein for the receptor for calcitonin generelated peptide (CGRP), a potent vasodilator neuropeptide. We have previously shown that this accessory protein, the CGRP-receptor component protein (RCP), is expressed in CGRP responsive tissues and that RCP protein expression correlates with the biological efficacy of CGRP in vivo. However, the function of RCP has remained elusive. In this study stable cell lines were made that express antisense RCP RNA, and CGRP-and adrenomedullin-mediated signal transduction were greatly reduced. However, the loss of RCP did not effect CGRP binding or receptor density, indicating that RCP did not behave as a chaperone but was instead coupling the CGRP receptor to downstream effectors. A candidate CGRP receptor named calcitonin receptor-like receptor (CRLR) has been identified, and in this study RCP co-immunoprecipitated with CRLR indicating that these two proteins interact directly. Since CGRP and adrenomedullin can both signal through CRLR, which has been previously shown to require a chaperone protein for function, we now propose that a functional CGRP or adrenomedullin receptor consists of at least three proteins: the receptor (CRLR), the chaperone protein (RAMP), and RCP that couples the receptor to the cellular signal transduction pathway.G protein-coupled receptors are generally thought to function as monomers that interact with G proteins to initiate signal transduction. However, it has recently been recognized that many G protein-coupled receptors require additional proteins for function. These proteins range from other receptors that form dimers, to heterologous accessory proteins that function primarily as chaperones (1, 2). In this study we report a novel accessory protein that does not act as a chaperone, but instead couples the receptor to the cellular signal transduction pathway. Thus, our concept of a G protein-coupled receptor involves a complex of proteins that are required for receptor function, including correct intracellular sorting, organization in the plasma membrane, and coupling to cellular signal transduction proteins.Calcitonin gene-related peptide (CGRP) 1 is a potent vasoactive neuropeptide, which has been implicated in vasodilation, migraine, and chronic pain (3-6). Despite the clinical implications of CGRP's biological actions, therapeutic strategies targeting CGRP have been hindered by the lack of a functional CGRP receptor. CGRP binding results in increased intracellular cAMP levels (7,8), and a candidate G protein-coupled receptor has been identified called the calcitonin receptor-like receptor (CRLR) (9). However, CRLR was initially non-functional when trans...

No abstract

. This observation provided an opportunity to map the determinants of receptor affinity exhibited by BIBN4096BS and the truncated analogs, Compounds 1 and 2. All three compounds exhibited higher affinity for the human receptor, human CRLR/human RAMP1, than for the rat receptor, rat CRLR/rat RAMP1. We have now demonstrated that this species selectivity was directed exclusively by RAMP1. By generating recombinant human/rat CRLR/RAMP1 receptors, we demonstrated that co-expression of human CRLR with rat RAMP1 produced rat receptor pharmacology, and vice versa. Moreover, with rat/human RAMP1 chimeras and site-directed mutants, we have identified a single amino acid at position 74 of RAMP1 that modulates the affinity of small molecule antagonists for CRLR/RAMP1. Replacement of lysine 74 in rat RAMP1 with tryptophan (the homologous amino acid in the human receptor) resulted in a >100-fold increase in antagonist affinities, similar to the K i values for the human receptor. These observations suggest that important determinants of small molecule antagonist affinity for the CGRP receptor reside within the extracellular region of RAMP1 and provide evidence that this receptor accessory protein may participate in antagonist binding.CGRP is a 37-amino acid neuropeptide that is expressed in a variety of cell types in both the central and peripheral nervous systems (5). In many tissues, CGRP-containing fibers are closely associated with blood vessels (6). Among the various physiological functions reported for CGRP, the most pronounced is vasodilation. CGRP is the most powerful of the vasodilator transmitters (7), and its vasoactive effects have been demonstrated in a variety of blood vessels (8), including those in the cerebral, coronary, and mesenteric vasculature.Mounting evidence suggests that CGRP is involved in the pathophysiology of migraine headache (9). Migraine is thought to be associated with dilation of cerebral blood vessels and activation of the trigeminovascular system (10). During the headache phase of a migraine, CGRP levels are elevated in the cranial circulation (11, 12). Successful treatment of the headache with sumatriptan resulted in normalization of CGRP levels (12), thus implicating CGRP in the pathophysiology of this disorder. Moreover, intravenous administration of CGRP to migraineurs induced a delayed migrainous headache in some patients (13). These observations suggest that inhibition of CGRP-mediated vasodilation may have therapeutic utility in the treatment of migraine.Research in the area of CGRP has intensified in recent years due in large part to the identification of its receptor, CRLR 1 (14), and the cloning of the receptor activity-modifying proteins (RAMPs) (1) and receptor component protein (15). CGRP activity is mediated by the G s -coupled G-protein-coupled receptor (GPCR), CRLR, which shares 55% homology with the calcitonin receptor. In an elegant study, McLatchie et al. (1) demonstrated that functional CGRP and adrenomedullin receptors are both derived from CRLR and that the phenotype i...

Calcitonin gene-related peptide and adrenomedullin belong to a structurally related neuropeptide family and are potent vasodilators expressed in the trigeminovascular system. The molecular identity of receptors for these proteins has only recently been elucidated. Central to functional binding of these neuropeptides is the G-protein-coupled receptor, the calcitonin receptor-like receptor (CRLR), whose cell surface expression and pharmacology is determined by coexpression of a receptor activity-modifying protein (RAMP). CRLR combined with RAMP binds calcitonin gene-related peptide with high affinity, whereas CRLR coexpression with RAMP2 or -3 confers high-affinity binding of adrenomedullin. The authors investigated the expression of these receptor components in human cerebral vasculature to further characterize neuropeptide receptor content and the potential functions of these receptors. Localization has been carried out using specific antisera raised against immunogenic peptide sequences that were subsequently applied using modern immunohistochemical techniques and confocal microscopy. The results are the first to show the presence of these receptor component proteins in human middle meningeal, middle cerebral, pial, and superficial temporal vessels, and confirm that both calcitonin gene-related peptide and adrenomedullin receptors may arise from the coassembly of RAMPs with CRLR in these vessel types. These novel data advance the understanding of the molecular function of the trigeminovascular system, its potential role in vascular headache disorders such as migraine, and may lead to possible ways in which future synthetic ligands may be applied to manage these disorders.