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The wide distribution of corticotrophin-releasing hormone (CRH) receptors in brain and periphery appear to be important in integrating the responses of the brain, endocrine and immune systems to physiological, psychological and immunological stimuli. The type 1 receptors are highly expressed throughout the cerebral cortex, a region involved in cognitive function and modulation of stress responses, where they are coupled to the adenylyl cyclase system. Using techniques that analyse receptor-mediated guanine-nucleotide binding protein (G-proteins) activation, we recently demonstrated that expressed type 1α CRH receptors are capable of activating multiple G-proteins, which suggests that CRH can regulate multiple signalling pathways. In an effort to characterize the intracellular signals generated by CRH in the rat cerebral cortex we sought to identify G-proteins activated by CRH in a physiological membrane environment. Rat cerebral cortical membrane suspensions were analysed for the ability of CRH to stimulate incorporation of [α-32P]-GTP-γ-azidoanilide to various G-protein α-chains. Our results show that CRH receptors are coupled to and activate at least five different G-proteins (Gs, Gi, Gq/11, Go and Gz) with subsequent stimulation of at least two intracellular signalling cascades. In addition, the photoaffinity experiments indicated that the CRH receptors preferentially activate the 45 kDa form of the Gsα-protein. This data may help elucidate the intracellular signalling pathways mediating the multiple actions of CRH especially under different physiological conditions.
Corticotropin-releasing hormone (CRH) is a 41-amino acid peptide (Vale et al. 1981), that is capable of integrating the neuroendocrine, behavioural, autonomic and immune responses to stress (Koob 1985; Besedovsky et al. 1986; Fisher 1989; Irwin 1993). CRH activates transcription of the proopiomelanocortin (POMC) gene and stimulates the release of ACTH and β endorphin from cells in the anterior pituitary gland. In addition, CRH can induce behavioural responses, stimulate thermogenesis, influence reproductive and cardiovascular function and exert both antiand pro-inflammatory effects (Chrousos et al. 1998; Fisher et al. 1982; Karalis et al. 1991). CRH is expressed in a number of brain regions including the hypothalamus, amygdala and cerebral cortex (Swanson et al. 1983), and receptors for CRH are distributed diversely throughout the brain.
The CRH receptor is a member of a specialized subfamily of GTP-binding protein (G-protein) coupled seven transmembrane domain (7TMD) receptors that includes the receptors for calcitonin, vasoactive intestinal peptide, and parathyroid hormone (PTH) (Perrin et al. 1993). Two subtypes of CRH receptors, termed R1 and R2, have been identified in the rat (Perrin et al. 1993; Lovenberg et al. 1995) and are encoded by separate genes. These two receptors share 70% identity. The R1 receptor binds CRH as well as CRH-like peptides (urocortin, urotensin and sauvagine) with equivalent high affinity, but the R2 receptor binds urocortin with higher affinity than the other CRH-like peptides, suggesting that urocortin may be the natural ligand (Vaughan et al. 1995). In the brain, the R1 receptor is found predominantly in the neocortex, cerebellum, olfactory bulb and anterior pituitary whilst the R2 receptor is localized to the subcortical, amygdaloid and hypothalamic regions (Primus et al. 1997); these data suggest that the R1 and R2 receptors might serve different roles in mediating CRH actions. Both the R1 and R2 genes encode multiple splice variants; the R2 gene exhibits even greater diversity through the use of alternative 5′ exons that produce three different receptors (R2α, R2β, R2γ).
Both R1 and R2 CRH receptors are coupled to Gs protein and activate adenylyl cyclase and cAMP production (Chen et al. 1986). Over the last few years it has been recognized that many members of the 7TMD receptor superfamily, including the PTH and the thyroid-stimulating hormone (TSH) receptor (Laugwitz et al. 1996; May and Gay 1997), are coupled to multiple G-proteins and thereby can regulate several signal transduction pathways. We have recently shown that the type 1α CRH receptor stably expressed in HEK293 cells can stimulate multiple G-proteins (Gs, Gi, Gq/11, and Go) (Grammatopoulos et al. 1999). These data are in agreement with other studies showing that intracellular signalling pathways other than adenylyl cyclase are activated by CRH such as the inositol triphosphate (IP3) and PKC pathways in Leydig cells, cultured astrocytes and epidermoid cells (Ulisse et al. 1990; Takuma et al. 1994; Kiang 1997). Also, in LLCPK-1 cells the stably expressed CRH-R1 is weakly coupled to the PLC-IP3 pathway (Nabhan et al. 1995). The rat cerebral cortex contains multiple CRH receptor isoforms (Grammatopoulos and Hillhouse 1998) that may be coupled to both cholera and pertussis toxin-sensitive G-proteins (Grammatopoulos et al. 1994). Accordingly, the present study was carried out to identify the G-proteins that are linked to rat cerebral cortical CRH receptors and to determine the mechanism by which the intracellular signals are generated.
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Considerable evidence suggests that CRH action in the brain is mediated via stimulation of the adenylyl cyclase system. The full complement of G-proteins and second messengers that can be activated by CRH have yet to be identified. This study, however, provides direct evidence that in the rat cerebral cortex, stimulation of endogenous CRH receptors leads to activation of multiple of G-protein subtypes with subsequent stimulation of at least two intracellular signalling cascades.
To identify G-proteins coupled to endogenous CRH receptors we used G-protein antisera to immunoprecipitate α subunits after incubation of membranes with 32P-GTP-AA and CRH. The reaction conditions for agonist-specific labelling of Gs-protein were dependent upon time and the concentration of CRH and GDP. Activation of Gs-proteins correlated with stimulation of adenylyl cyclase activity and generation of cAMP. Interestingly, incorporation of 32P-GTP-AA to Gs-protein occurred at a 10-fold lower concentration of CRH than required for stimulation of adenylyl cyclase. Although this discrepancy might be due to experimental conditions or to the sensitivity of the cAMP assay, it is similar to the kinetic relationship between receptor binding and activation of adenylyl cyclases that has led to the concept of spare receptors. Whether our findings imply that a threshold number of Gs molecules are required to induce activation of adenylyl cyclase or that some CRH-coupled Gs molecules are not able to activate adenylyl cyclase (e.g. due to differences in membrane distribution) cannot be determined from these experiments. Immunoblot analysis showed that cerebral cortical membrane homogenates contain substantially more of the 52 KDa isoform of Gsα than of the short form of Gs. Surprisingly our 32P-GTP-AA photoaffinity experiments indicated that the CRH receptors preferentially activate the 45 kDa form of the Gsα-protein. However, this appears to be a tissue specific phenomenon since using the same labelling procedure in human myometrium, CRH activated both 45 and 52 kDa forms with equal potency (Grammatopoulos & Hillhouse, unpublished observations).
Our results also demonstrated for the first time that the CRH receptors in the rat cerebral cortex are coupled to multiple G-proteins, including Gs, Gq/11, Gi1/2, Go and Gz. This finding is in agreement with our previous experiments which demonstrated that the rat cerebral cortex CRH receptors are coupled to cholera and pertussis toxin-sensitive G-proteins (Grammatopoulos et al. 1994), a finding that is true for many members of the 7TMD receptors family. This property of the CRH receptor has also been demonstrated in HEK293 cells stably expressing the CRH receptor type 1α (May and Gay 1997). Activation of all the G-proteins occurred at subnanomolar concentrations of CRH except the Gi1/2- and Gz-proteins, which suggests that the CRH-R is relatively weakly coupled to these G-proteins. Western blot analysis showed that all types of G-proteins were present in the cerebral cortical membrane homogenates (data not shown). CRH-induced activation of these proteins could be blocked by the peptide CRH receptor antagonist astressin, whilst urocortin, the other CRH-R1 ligand, had comparable G-protein activation profile and potency. Urocortin was more potent than CRH in its ability to activate Gq-proteins, and the functional importance of this interaction requires further investigation.
We confirmed the specificity of our techniques by using agonists known to activate specific G-proteins such as α- and β-adrenergic receptor agonists. Furthermore, PTX-pretreatment of the rat cortical membranes abolished the CRH-R-induced activation of the Gi and Go-proteins, consistent with the view that PTX-stimulated ADP-ribosylation retains the G-protein in its heterotrimeric GDP-bound inactive form, therefore reducing the incorporation of GTP (or GTP-AA) (Moss and Vaughan 1988). Moreover, PTX-pretreatment had no effect on the 32P-GTP-AA labelling of Gsα or Gq/11α, α chains that are not PTX-substrates.
Previous studies had demonstrated that CRH receptors in rat Leydig cells are coupled to PTX-insensitive G-proteins and activate phospholipase C but not adenylyl cyclase (Ulisse et al. 1990). Our 32P-GTP-AA labelling experiments confirmed these earlier observations, and showed that CRH could stimulate increased GTP-AA incorporation in Gq/11- and Gi-, but not Gs-proteins. By contrast, hCG, acting through the LH receptor, induced 32P-GTP-AA labelling of Gsα. In these cells the CRH receptor does not appear to couple to adenylyl cyclase, a phenomenon that is similar to that found in human placenta and fetal membranes (Karteris et al. 2000).
Here we showed that in rat cerebral cortical membranes activation of Gq/11, Gi and Go G-proteins by CRH could stimulate phospholipase C with subsequent generation of inositol triphosphates suggesting the existence of an alternative second messenger pathway by which CRH can exert its actions. It is also possible that CRH-induced activation of Gi-proteins may lead to liberation of βγ subunits which in turn can activate types II/IV/VII adenylyl cyclase (Ahmed and Heppel 1997) all of which are present in the cerebral cortex (Hellevuo et al. 1996; Mons et al. 1998). Such synergistic interactions between Gs and Gi-dependent signalling pathways in the stimulation of type II adenylate cyclase have been well-documented (Tsu and Wong 1996) and would explain the partial inhibition of the basal and CRH-stimulated adenylate cyclase activity observed following PTX treatment of cerebral cortical membranes (Grammatopoulos et al. 1994).
Also, our experiments demonstrated for the first time that the CRH receptor can activate Go- and Gz-proteins. These G-proteins have been implicated in the inhibition of adenylase cyclase and modulation of cation channels (Harris-Warrick et al. 1988; Fields and Casey 1997); however, thus far the physiological consequences of their activation remain unknown.
In summary, we have demonstrated that multiple G-proteins are coupled to CRH receptors in the rat cerebral cortex. Because these investigations were carried out in a physiological membrane system, the G-protein/CRH receptor interactions described are believed to be representative of natural associations between these proteins. Our previous isoelectric focusing data suggested that the rat cerebral cortex contains at least three different CRH receptor types (Grammatopoulos and Hillhouse 1998) which belong to the type R1 CRH receptor family (Grammatopoulos, Phull & Hillhouse, unpublished observations) and might represent differentially glycosylated or other post-translational modification products of the CRH-R1. It is possible that the different CRH-R1 isoforms are coupled to different G-proteins. Although it is possible that post-translational modification can modify coupling of the receptor to specific G-proteins, at present we do not know whether each of the three CRH-R1 receptor isoforms are coupled to unique G-proteins and or all receptor isoforms are able to activate several G-proteins and the mechanism of coupling specificity is currently under investigation.