The corticotropin-releasing factor (CRF) 3 receptor family is involved in the regulation of the hypothalamic-pituitary-adrenal stress axis in mammals (1-3). A large body of evidence points to a major role of the receptors in mediating CRF effects in anxiety and depressive disorders and in stress-associated pathologies. Two types of CRF receptors are known, the CRF 1 and the CRF 2 receptors. The CRF 1 receptor is expressed mainly in the pituitary and central nervous system and binds CRF with high affinity. It mediates adrenocorticotrophic hormone release from the anterior pituitary and is involved in the endocrine, autonomic, and cognitive responses to stress stimuli. The CRF 2 receptors are expressed in the central nervous system but also in the periphery including skeletal muscle cells, cardiac myocytes, and cells of the gastrointestinal tract. Three splice variants of CRF 2 receptors have been described: CRF 2(a) , CRF 2(b) , and CRF 2(c) receptors. They bind CRF with low and the urocortins 1-3 with high affinity. The CRF 2 receptors are involved in the regulation of feeding behavior (4) and in recovery from a stress response (5). It is likely that they are also involved in modulating anxiety-related behavior.The CRF receptors belong to the small subgroup of GPCRs (5-10%) possessing putative N-terminal signal peptides. These peptides are believed to be cleaved-off after mediating the ER targeting/insertion process (6, 7). The majority (90 -

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Approximately 5-10% of the GPCRs (G-protein-coupled receptors) contain N-terminal signal peptides that are cleaved off during receptor insertion into the ER (endoplasmic reticulum) membrane by the signal peptidases of the ER. The reason as to why only a subset of GPCRs requires these additional signal peptides is not known. We have recently shown that the signal peptide of the human ET(B)-R (endothelin B receptor) does not influence receptor expression but is necessary for the translocation of the receptor's N-tail across the ER membrane and thus for the establishment of a functional receptor [Köchl, Alken, Rutz, Krause, Oksche, Rosenthal and Schülein (2002) J. Biol. Chem. 277, 16131-16138]. In the present study, we show that the signal peptide of the rat CRF-R1 (corticotropin-releasing factor receptor 1) has a different function: a mutant of the CRF-R1 lacking the signal peptide was functional and displayed wild-type properties with respect to ligand binding and activation of adenylate cyclase. However, immunoblot analysis and confocal laser scanning microscopy revealed that the mutant receptor was expressed at 10-fold lower levels than the wild-type receptor. Northern-blot and in vitro transcription translation analyses precluded the possibility that the reduced receptor expression is due to decreased transcription or translation levels. Thus the signal peptide of the CRF-R1 promotes an early step of receptor biogenesis, such as targeting of the nascent chain to the ER membrane and/or the gating of the protein-conducting translocon of the ER membrane.

The corticotropin-releasing factor receptor type 2a (CRF 2(a) R) belongs to the family of G protein-coupled receptors. The receptor possesses an N-terminal pseudo signal peptide that is unable to mediate targeting of the nascent chain to the endoplasmic reticulum membrane during early receptor biogenesis. The pseudo signal peptide remains uncleaved and consequently forms an additional hydrophobic receptor domain with unknown function that is unique within the large G protein-coupled receptor protein family. Here, we have analyzed the functional significance of this domain in comparison with the conventional signal peptide of the homologous corticotropin-releasing factor receptor type 1 (CRF 1 R). We show that the presence of the pseudo signal peptide leads to a very low cell surface receptor expression of the CRF 2(a) R in comparison with the CRF 1 R. Moreover, whereas the presence of the pseudo signal peptide did not affect coupling to the G s protein, G i -mediated inhibition of adenylyl cyclase activity was abolished. The properties mediated by the pseudo signal peptide were entirely transferable to the CRF 1 R in signal peptide exchange experiments. Taken together, our results show that signal peptides do not only influence early protein biogenesis. In the case of the corticotropin-releasing factor receptor subtypes, the use of conventional and pseudo signal peptides have an unexpected influence on signal transduction.The family of corticotropin-releasing factor (CRF) 3 receptors encompasses two subtypes, the CRF 1 R and CRF 2 R (1, 2). The CRF 1 R is expressed mainly in the anterior pituitary and plays a central role in the regulation of the hypothalamic-pituitaryadrenal stress axis in mammals (3). It binds CRF with high affinity and mediates ACTH release from the pituitary leading to cortisol biosynthesis in the adrenal cortex. A large body of evidence points to a major role of the receptor in mediating CRF effects in anxiety and depressive disorders (4 -6).In the case of the CRF 2 R, three splice variants have been described as follows: the CRF 2(a) R, CRF 2(b) R, and CRF 2(c) R. All splice variants bind CRF with low affinity and the urocortins 1-3 with high affinity. They are involved in the regulation of feeding behavior (7) and in recovery from a stress response (8).It is likely that they are also involved in modulating anxietyrelated behavior.Both the CRF 1 R and the CRF 2(a) R usually couple to the G s / adenylyl cyclase system and consequently increase cytosolic cAMP as a second messenger. However, a promiscuous coupling behavior was described previously in particular for the CRF

The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.