Kevin R. Oliver

CGRP-RCP, a Novel Protein Required for Signal Transduction at Calcitonin Gene-related Peptide and Adrenomedullin Receptors

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 gene-related 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.

Composition of perineuronal nets in the adult rat cerebellum and the cellular origin of their components

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. J. Comp. Neurol. 494:559–577, 2006.

Immunohistochemical localization of calcitonin receptor-like receptor and receptor activity-modifying proteins in the human cerebral vasculature

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 RAMP1 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.

Localization of 5-ht5A receptor-like immunoreactivity in the rat brain

5-Hydroxytryptamine exerts modulatory physiological effects on both the central and peripheral nervous systems by activation of discrete receptor families. 5-ht(5A) is among the recently cloned, novel 5-HT receptors and currently under investigation to identify its pharmacological characteristics and potential physiological function(s). In this study, antibodies raised to a 5-ht(5A)-specific peptide were characterized using dot blot, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunohistochemistry, and the distribution of 5-ht(5A)-like immunoreactive material determined in rat brain. A major band of 41 kDa was observed following SDS-PAGE, corresponding to the predicted size of this receptor. 5-ht(5A)-like immunoreactivity was detected in areas known to have significant serotonergic input, such as hypothalamic and amygdaloid nuclei. Interestingly, 5-ht(5A)-like immunoreactivity showed a predilection for the suprachiasmatic nucleus, suggesting its possible role in the regulation of circadian rhythms, a function previously ascribed to 5-HT(1A) and 5-HT(7) receptors.

Distribution of 5-ht5A, 5-ht5B, 5-ht6 and 5-HT7 receptor mRNAs in the rat brain

The precise involvement of 5-ht(5A), 5-ht(5B), 5-ht(6) and 5-HT(7) receptors in the pleiotropic actions of 5-HT remain incompletely known. To gain insights into their physiological function(s), localization of mRNAs encoding these subtypes was carried out using in situ hybridization on rat brain sections. Localization was heterogeneous. For example, 5-ht(5A) mRNA was widely expressed while 5-ht(5B) mRNA was predominantly expressed in habenula, hippocampus and inferior olive. 5-ht(6) mRNA was abundant in olfactory tubercles and caudate putamen, and highest levels of 5-HT(7) mRNA were observed in multiple thalamic nuclei. These data suggest that these receptors may have distinct functional roles within the serotonergic system.

The role of the CGRP-receptor component protein (RCP) in adrenomedullin receptor signal transduction

G protein-coupled receptors are usually thought to act as monomer receptors that bind ligand and then interact with G proteins to initiate signal transduction. In this study we report an intracellular peripheral membrane protein named the calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) required for signal transduction at the G protein-coupled receptor for adrenomedullin. Cell lines were made that expressed an antisense construct of the RCP cDNA, and in these cells diminished RCP expression correlated with loss of adrenomedullin signal transduction. In contrast, loss of RCP did not diminish receptor density or affinity, therefore RCP does not appear to act as a chaperone protein. Instead, RCP represents a novel class of protein required to couple the adrenomedullin receptor to the cellular signal transduction pathway. A candidate adrenomedullin receptor named the calcitonin receptor-like receptor (CRLR) has been described, which forms high affinity adrenomedullin receptors when co-expressed with the accessory protein receptor-activity modifying protein 2 (RAMP2). RCP co-immunoprecipitated with CRLR and RAMP2, indicating that a functional adrenomedullin receptor is composed of at least three proteins: the ligand binding protein (CRLR), an accessory protein (RAMP2), and a coupling protein for signal transduction (RCP).

Distribution of novel CGRP1 receptor and adrenomedullin receptor mRNAs in the rat central nervous system

Recent cloning studies have isolated receptors which confer specific responsiveness to calcitonin gene related peptide (CGRP) and the related peptide adrenomedullin. Using in situ hybridisation, we demonstrate the heterogenous distribution of the mRNAs of two proposed CGRP1 receptors (RDC-1 and calcitonin receptor-like receptor, CRLR) in the rat brain. Adrenomedullin receptor mRNA was weakly expressed, principally in the cerebellum. These findings may assist in the determination of the function of these largely uncharacterised receptors.

Discrete expression of TRPV2 within the hypothalamo-neurohypophysial system: Implications for regulatory activity within the hypothalamic-pituitary-adrenal axis

Transient receptor potential channel proteins (TRPs) constitute a steadily growing family of ion channels with a range of purported functions. It has been demonstrated that TRPV2 is activated by moderate thermal stimuli and, in the rat, is expressed in medium to large diameter dorsal root ganglion neurons. In this study, antisera specific for the human TRPV2 homologue were raised and characterized for immunohistochemical use. Subsequently, thorough investigation was made of the localization of this cation channel in the macaque primate brain. TRPV2‐immunoreactive material was highly restrictively localized to hypothalamic paraventricular, suprachiasmatic, and supraoptic nuclei. Confocal double‐ and triple‐labeling studies demonstrated that TRPV2 immunoreactivity is preferentially localized to oxytocinergic and vasopressinergic neurons. Few, if any, cells in these regions expressed TRPV2 immunoreactivity in the absence of oxytocin immunoreactivity or vasopressin immunoreactivity. Expression in the paraventricular and supraoptic nuclei suggests that TRPV2 is likely to play a fundamental role in mediating cation transport in neurohypophysial neurons. TRPV2 has been shown to be translocated upon cell activation and neurons expressing TRPV2 immunoreactivity in vivo are among those known to engage in sporadic, intense activity. Taken together, these data suggest that this channel may play a vital role in mediating physiological activities associated with oxytocin and vasopressin release such as parturition, lactation, and diuresis. These data may also implicate the involvement of TRPV2 in disorders of the hypothalamic‐pituitary‐adrenal axis, including anxiety, depression, hypertension, and preterm labor. J. Comp. Neurol. 474:24–42, 2004.

Autoradiographic localization of 125I[Tyr14] nociceptin/ orphanin FQ binding sites in macaque primate CNS

Nociceptin/orphanin FQ (N/OFQ) is a recently identified neuropeptide that has been implicated in a multitude of CNS functions. These include nociception, feeding, cognition, locomotion, stress and neuroendocrine control. The endogenous receptor for this ligand is the nociceptin/orphanin FQ peptide (NOP) receptor. The distribution of NOP in rodent has been widely reported by the use of in situ hybridization, immunohistochemistry and autoradiographic radioligand binding but less is known of its localization in higher species. We have therefore sought to optimize and determine the distribution of (125)I[Tyr(14)]N/OFQ binding sites in macaque primate brain and spinal cord. Highest levels of binding were observed in neocortical areas, hippocampus, amygdala, caudate nucleus and putamen, medial thalamic nuclei and superficial laminae of the superior colliculus. These novel data present for the first time, the distribution of N/OFQ receptors in non-human primate CNS and, by comparison with localization in the rat, reveal that species differences may exist in the distribution of this neuropeptide receptor. These data have important implications regarding the roles of N/OFQ across species and may have ramifications in the interpretation of preclinical pharmacological studies.

Quantitative Comparison of Pretreatment Regimens Used to Sensitize In Situ Hybridization Using Oligonucleotide Probes on Paraffin-embedded Brain Tissue

Paraffin embedding of tissue is generally perceived to dramatically reduce RNA detectability. As a consequence, in situ hybridization on paraffin-embedded tissue is largely confined to detection of high-copy RNA species (e.g., viral RNA) and/or to detection using typically more sensitive cDNA probes or riboprobes. In this study, several procedures for in situ hybridization on paraffin-embedded rat tissue using oligonucleotide probes complementary to cellular transcripts were developed and quantitatively compared. Certain pretreatments showed marked increases in sensitivity compared to untreated sections. Furthermore, through quantitative assessment using image analysis, sensitivity of optimal pretreatments was equal to that of routinely used fresh-frozen, postfixed tissue sections. The development of such techniques permitting in situ hybridization to be carried out on paraffin-embedded tissue allows a comparison of protein and mRNA distribution to be made in adjacent sections and provides the potential for double labeling by in situ hybridization and immunohistochemistry which may not be possible on post-fixed frozen sections.