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...

DAXX, a modulator of apoptosis and a repressor of basal transcription, was identified in a two-hybrid screen as a protein capable of interacting with a trimeric form of human heat shock factor 1 (HSF1). In human cells, DAXX interacted with HSF1 essentially only during stress, i.e., when factor trimerization occurred. Several lines of experimentation suggested that DAXX is an important mediator of HSF1 activation: (i) overexpression of DAXX enhanced basal transactivation competence of HSF1 in the absence of a stress; (ii) a DAXX fragment exerted dominant-negative effects on HSF1 activation by different types of stress; (iii) induction of heat shock or stress protein (HSP)70 by heat stress was defective in a cell line lacking functional DAXX; and (iv) RNA interference depletion of DAXX also substantially reduced heat induction of HSF1 activity and HSP70 expression. HSF1 transactivation competence is repressed by an HSP90-containing multichaperone complex that interacts with trimeric factor. Overexpressed HSF1, known to be largely trimeric, only marginally increased HSF1 activity on its own but potentiated the activating effect of DAXX overexpression. Expression of a nonnative protein capable of competing for multichaperone complex also synergistically enhanced activation of HSF1 by DAXX. These observations suggest a model in which DAXX released from its nuclear stores during stress opposes repression of HSF1 transactivation competence by multichaperone complex through its interaction with trimerized HSF1. Our identification of DAXX as a mediator of HSF1 activation raises the question whether DAXX produces some of its pleiotropic effects through modulation of HSP levels.

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