Emerging role of alternative splicing of CRF 1 receptor in CRF signaling

Alternative splicing of mRNA is one of the most important mechanisms responsible for an increase of the genomic capacity. Thus the majority of human proteins including G protein-coupled receptors (GPCRs) possess several isoforms as a result of mRNA splicing. The corticotropin-releasing factor (CRF) and its receptors are the most proximal elements of hypothalamic-pituitaryadrenal axis (HPA) — the central machinery of stress response. Moreover, expression of CRF and regulated activity of CRF receptor type 1 (CRF1) can also play an important role in regulation of local stress response in peripheral tissues including skin, gastrointestinal tract or reproductive system. In humans, expression of at least eight variants of CRF1 mRNA (α, β, c, d, e, f, g and h) was detected and alternative splicing was found to be regulated by diverse physiological and pathological factors including: growth conditions, onset of labor, during pregnancy or exposure to ultraviolet irradiation. The pattern of expression of CRF1 isoforms is cell type specific and recently has been linked to observed differences in responsiveness to CRF stimulation. In the proposed model of regulation of CRF-signaling, isoform CRF1α plays a central role. Other isoforms modulate its activity by oligomerization, leading to alteration in receptor trafficking, localization and function. Co-expression of CRF1 isoforms modulates sensitivity of cells to the ligands and influences downstream coupling to G-proteins. The other possible regulatory mechanisms include fast mRNA and/ or protein turnover or decoy receptor function of CRF1 isoforms. Taken together, alternative splicing of CRF1 can represent another level of regulation of CRF-mediated stress responses at the central and peripheral levels. Chronic stress or malfunction of the HPA-axis have been linked to numerous human pathologies, suggesting that alternative splicing of CRF1 receptor could represent a promising target for drugs development.

The peripheral expression of CRF, its receptors and other elements of the HPA axis are crucial in locally targeted regulation of the stress response, which is important in organs constantly subjected to stress insults like the skin (Slominski et al., 1999;2000b;Slominski & Wortsman, 2000;Arck et al., 2006).

CRF receptors
Human and all vertebrates express two types of CRF receptor -CRF1 and CRF2, also named: CRH-R1 and CRH-R2, or CRFR1 and CRFR2 (Perrin & Vale, 1999;Hauger et al., 2006;Hillhouse & Grammatopoulos, 2006;Slominski et al., 2006b).In the catfish a third member of the CRF receptor family was described as CRF3, which appears to be unique for this species (Arai et al., 2001).The CRF1 and CRF2 share about 70 % of homology among different species and CRF3 shows higher homology to CRF1 than CRF2.CRF receptors seems to have originated from one ancestor gene, which was duplicated (in the case of the catfish triplicated) in order to differentiate responsiveness to the selected ligands.Accordingly, CRF1 has high affinity for CRF and UCN-I, and very low for UCN-II, while CRF2 possesses high affinity for UCN-I, UCN-II and a lower one for CRF (Dautzenberg et al., 2001;Hillhouse et al., 2002;Bale & Vale, 2004;Hemley et al., 2007).CRF3 bound CRF with a 5-fold higher affinity than urotensin-I and sauvagine (Arai et al., 2001).
Although activation of CRF receptors is predominantly linked to up-regulation of cAMP through G sa subunit and adenylate cyclase pathway, the phenotypic outcome is different depending on anatomic location or cell type.On the central level, stimulation of CRF1 results in production of ACTH leading to arousal, somatic nervous system activation and under chronic conditions causes depression.On the other hand, activation of CRF2 leads to appetite suppression and acts like an antidepressant.Thus, CRF1 is implicated in normal response to stress, while CRF2 meditates fine-tuning of the stress response.Moreover, two CRF receptors have opposite functions in regulation of behavior with CRF1 mediating anxiogenic and CRF2 anxiolytic effects (Hillhouse et al., 2002;Bale & Vale, 2004).
The general structure of CRF receptors is similar to other GPCRs and consists of an extracellular domain (eCD) or ligand binding domain encompassing the Nterminal fragment and three extracellular coils (eC1-3); a seven α-helical transmembrane domain (7TM) including three internal coils (IC1-3) and the C-terminus (Fig. 2  and 3).The most variable region of the CRF receptors is the N-terminal eCD with only 40 % of homology between CRF1 and CRF2.The rest of the sequence is highly conserved with homology of 80 % or greater for the 7TM domain and intracellular and extracellular loops.The third IC responsible for interaction with G-proteins was found to be identical for all CRFs (Hillhouse et al., 2002;Hemley et al., 2007).

Alternative splicing of CRF1 mRNA
In human, expression of at least eight splicing variants of CRF1 has been detected so far, and several other isoforms have been detected in other organisms or could be theoretically predicted (Fig. 2) (Pisarchik & Slominski, 2001;2002;Hillhouse & Grammatopoulos, 2006;Slominski et al., 2006b;2007b).
Only one isoform, CRF1β, also called pro-CRH-R1 (Teli et al., 2008) is coded by all 14 exon, because in the sequence of other isoforms exon 6 is spliced out.CRF1 splicing variants could be divided into three groups: full-length receptors (CRF1α, β), C-terminal, or eCD mutants (CRF1c and e) and isoforms with an impaired or missing 7TM domain (CRF1d, e, f, g and h).So-called "soluble" receptors (CRF1e, h) form a special subgroup of 7TM mutants lacking the entire 7TM domain.The main isoform CRF1α represents fully functional receptor and CRF1c has exon 3 spliced out.In isoform CRF1d exon 13 is missing.In the sequence of CRF1e exons: 3 and 4 are excluded, which causes a frameshift and introduction of a premature stop codon.CRF1f has exon 12 removed and in the sequence of CRF1g 27 base pairs of exon 10, the whole exon 11 and 28 base pairs of exon 12 are absent.CRF1h has an insertion of a cryptic exon between exons 4 and 5, resulting in a premature termination due to a frameshift (Pisarchik & Slominski, 2001;2002;Slominski et al., 2006b).In addition, mRNA of four unique isoforms (j, k, m and n) was detected in hamster (Pisarchik & Slominski, 2002).Isoform CRF1j has a deletion of exon 5 resulting in a frameshift in exon 6 (in rodents CRF1 has only 13 exons and there is no equivalent of human exon 6).Other isoforms named CRF1k, m and n have deletions within the 7TM domain as follows: exon 10; exon 11 and 12; or exons 10 to 12, respectively (Pisarchik & Slominski, 2002).
Recent studies have suggested that alternative splicing of CRF1 might be independent from regulation of receptor expression (Markovic et al., 2007).Thus it can be proposed that splicing changes the pool of CRF1α mRNA.Interestingly, an induction of CRF1 splicing by cAMP might suggest the presence of a negative feedback, where an elevated concentration of cAMP due to stimulation of the CRF1α receptor would result in alternative splicing of the receptor, leading to at least partial inhibition of CRF signaling.

ReGulATION OF CRF sIGNAlING by CRF1 IsOFORMs
Structure-function relationship plays a very important role in determination of activities of GPCRs and their isoforms.Detailed studies on CRF1 mutants and splicing variants revealed the functional importance for eCD and 7TM domains including external and internal coil regions and the C-terminus (Sydow et al., 1997;Hofmann et al., 2001;Perrin et al., 2001;Hillhouse & Grammatopoulos, 2006;Slominski et al., 2006b;Markovic et al., 2008;Zmijewski & Slominski, 2009a).As predicted, expression of eCD, alone, was sufficient for ligand binding, but the binding was enhanced by the presence of extracellular loops (Sydow et al., 1997).The 7TM domain is essential for proper signal transduction, resulting in activation of G-proteins and subsequent production of second messengers (cAMP, IP 3 and Ca +2 ).IC-3 was shown to be involved in G-protein interaction and the presence of the C-terminus enhanced cAMP production (Grammatopoulos et al., 1999;Pisarchik & Slominski, 2004).In addition to structural properties of CRF1 isoforms (and other GPCRs), their activity depends on their cellular localization.Specifically, CRF1 isoforms can be divided into three categories: membrane-bound, intracellular and extracellular (soluble) receptor forms (Slominski et al., 2006b).
It should be noted that a change in the receptor sequence by alternative splicing often results in frameshift and is responsible for premature termination of transcription.Such transcripts are prone to activate the nonsense-mediated mRNA decay mechanism (Lewis et al., 2003).Nevertheless, recent advances in receptor physiology suggest that these mRNA might encode proteins with modified but functional properties (Pisarchik & Slominski, 2004;Hillhouse & Grammatopoulos, 2006;Slominski et al., 2006b;Zmijewski & Slominski, 2009a;2009b).

Classical pathway -CRF1α
CRF1α is the main and fully functional isoform of the receptor and the majority of publications concerning the CRF1 receptor (CRFR1, CRHR1, CRH-R1) refer to this isoform.
In the classical pathway, ligand binding to CRF1 leads to signal transduction across the cell membrane resulting in activation of heterotrimeric G-proteins (Fig. 4).Detailed studies have revealed that the CRF receptor interacts with several G-proteins with preference for G αs and lower affinity for G αo , G αq/11 , G αi and G αz (Grammatopoulos et al., 1999).Although, the primary target is represented by the activation of G αs (adenylate cyclase (AC) -protein kinase A (PKA) pathway), it has to be noted that activation of specific G-proteins and signaling pathways can be tissue and cell type specific (Slominski et al., 2006a).It remains to be investigated if expression of alternative isoforms of CRF1 is responsible for modulation of affinity for G-proteins or whether this process is regulated by other mechanisms including, e.g., phosphorylation of the receptor.
Thus, the activation of secondary messengers and phenotypic effects of CRF1α stimulation is cell type-dependent and regulated by external and internal factors.It is tempting to conclude that such a diversity is regulated at least in part by expression of several CRF1 isoforms.Moreover, recent studies have revealed that alternative splicing is a dynamic process of adaptation to the changing environment (Markovic et al., 2007;Zmijewski & Slominski, 2009a).Unfortunately, there is a shortage of information on the phenotypic consequences of alternative splicing of CRF1 which might lead to wrong assumption concerning CRF signaling in given cellular model.

Membrane-bound isoform -CRF1β
CRF1β is sometimes referred to as pro-CRF1, because it is encoded by all the 14 exons, including exon 6 spliced out from all the other known isoforms (Chen et al., 1993;Xiong et al., 1995).Integration of the 29-amino-acid fragment encoded by exon 6 into first intracellular coil of CRF1 only slightly inhibits substrate-binding properties of the receptor (2.1×), but cAMP production was found to be impaired (100× decrease) (Xiong et al., 1995).Recent studies have shown that a positively charged fragment (F170-R174) of the CRF1β insert is responsible for inhibition of cAMP response (Teli et al., 2008).Furthermore, retention of exon 6 also increases responsiveness to PKC-induced phosphorylation and results in premature desensitization of signaling and internalization of the receptor (Markovic et al., 2006).Thus, despite of proper cell membrane localization CRF1β and possession of all the structural features of the native receptor, CRF signaling through CRF1β is impaired.

Membrane bound isoform -CRF1c
CRF1c, the other example of a CRF1 isoform possesses an intact 7TM domain (Ross et al., 1994), which indicates proper cell membrane localization (Slominski et al., 2006b), and this has been recently confirmed experimentally (Zmijewski & Slominski, 2009a;2009b).On the other hand, a deletion of 40 amino acids encoded by exon 3 removes the central part of eCD including Ligand binding to CRF1 receptor activates at least three G α subunits (α s , α i and α q ) of G-protein resulting in stimulation cAMP production by adenylate cyclase (AC) with subsequent cAMP-induced phosphorylation of CREB and activation of the cAMP-responsive element (CRE).Mobilization of calcium (Ca +2 ) results in activation of the calcium-responsive element (CARE).Activation of AP-1 can also trigger the mitogen-activated protein kinase (MAPK) pathway through protein kinase A (PKA).In addition, α q -mediated stimulation of phospholipase C results in IP 3 -driven activation of activator protein 1 (AP-1) dependent promoters.Downstream signaling from CRF1 receptor regulates expression of several genes including POMC, several interleukins, involucrine and cytokeratin 14.The phenotypic effects of stimulation include: stimulation of differentiation, steroidogenesis, melanogenesis and release of arachidonic acid.CRF inhibits cell division and regulates immune response.See text and citations within for details.Alternative splicing of CRF1 three out of six cysteines.These residues are crucial in formation of disulfide bridges essential for stability of the eCD domain (Fig. 3).Thus, in spite of the proper membrane localization, CRF1c expressed in the kidney cell line COS-1, failed to bind agonists ( 125 I-oCRF) (Ross et al., 1994).elevation of the intracellular level of cAMP was observed only after stimulation with a high concentration of human CRF (Ross et al., 1994;Grammatopoulos et al., 2000).It has to be noted that detection of CRF stimulated accumulation of cAMP in cells overexpressing CRF1c could be at least partially explained by endogenous expression of CRF1 shown in the another fibroblast-like kidney cell line COS-7 (Slominski et al., 2007b).Moreover, CRF and urocortin were not able to stimulate CRF1c mediated IP 3 production, MAPK phosphorylation or CRe activation in human embryonic kidney line (HeK 293) overexpressing CRF1c (Grammatopoulos et al., 2000;Markovic et al., 2008).
CRF1d with a characteristic deletion of a 14-amino-acid fragment of the 7TM domain coded by exon 12, was first described by the Gramatopoulos group (Grammatopoulos et al., 1999;Hillhouse & Grammatopoulos, 2006).The deletion of a fragment of the seventh transmembrane α-helix did not result in a decrease in substrate binding affinity, when compared with CRF1α.However, distortion of the 7TM domain inhibits interaction of the receptor with G-proteins (G αs , G αo , G αq and G αi ) and results in subsequent inhibition of downstream signaling, as judged by an at least 10-fold decrease in cAMP synthesis and total inhibition of IP 3 production (Grammatopoulos et al., 1999).As a consequence, inhibition of activation of cAMP or IP 3 response elements (CRe and AP-1) was observed in cells overexpressing CRF1d (Zmijewski & Slominski, 2009a;2009b).In SKMeL-188 melanoma cells, which only express the CRF1d isoform, the lack of coupling to cAMP with overt stimulation of Ca 2+ flux suggest that the CRF1d signal transduction pathway is only coupled to either voltage-activated Ca 2+ ion channels or PLC (Slominski et al., 2006a;Zmijewski & Slominski, 2009b).Ovine CRF1var was also shown to bind oCRF (ovine CRF) with comparable affinity to the native receptor, but oCRF-stimulated accumulation of cAMP and internalization of the receptor was impaired (Myers et al., 1998).Inhibition of downstream signaling as measured by cAMP accumulation or activation of CRe, CARe, AP-1 transcription elements was also observed in various cells overexpressing human isoforms CRF1f and g (Pisarchik & Slominski, 2004;Zmijewski & Slominski, 2009a;2009b).Thus, it is postulated that distortion within the 7TM domain resulting from alternative splicing of CRF1 mRNA does not significantly influence an affinity for ligands, but affects binding to Gprotein and, consequently downstream signaling through the receptor is impaired.
Recent studies have shown that overexpression of CRF1d in contrast to CRF1α resulted in intracellular accumulation of this 7TM mutant (Markovic et al., 2008;Zmijewski & Slominski, 2009a;2009b).Intracellularly, CRF1d was found to be associated with the endoplasmic reticulum and CRF1f and g with Golgi cisterns in human HaCaT keratinocytes (Zmijewski & Slominski, 2009a).These findings might explain high intracellular CRF1 immunoreactivity in human skin tissue and cell lines or uterine smooth muscle cells, known to express multiple CRF1 isoforms including 7TM mutants (Slominski et al., 2006a;Markovic et al., 2007;Zmijewski & Slominski, 2009a).
A recent study by Markovic and collaborators (Markovic et al., 2008) showed that cassette G356-F358 within the seventh transmembrane helix of CRF1α (exon 13) is crucial for membrane localization of the receptor (Figs. 2 and 3).Deletion or misplacement of this fragment is characteristic for all 7TM isoforms of CRF1 (Figs. 2 and 3).Interestingly, CRF1d and a calcitonin receptor splicing variant (CRTDe13) have an analogous exclusion of exon 13 with similar physiological consequences (Shyu et al., 1996;Seck et al., 2003;2005).Moreover, co-expression of CRF1α with CRF1d, f or g results in retention of both isoforms inside the cell (Zmijewski & Slominski, 2009a;2009b).Intracellular retention of CRF1 isoforms is most likely associated with their oligomerization (Kraetke et al., 2005;Mikhailova et al., 2007;Markovic et al., 2008;Zmijewski & Slominski, 2009a;2009b).Thus, it could be postulated that distortion of the 7TM domain of CRF1 receptor (and other GPCRs) results in at least partial retention of the reception in the cell, impairs G-protein binding and signal transduction despite only slightly altered ligand binding.In addition an intracellular localization sequesters the receptor and its homo-and heterodimers prevents an interaction with extracellular substrates.
CRF1h (human, mouse and hamster) similarly to CRF1j (only found in hamster) are encoded by exons 1-4 of full length CRF1.Deletion of exon 5 results in a frameshift and introduction of a premature stop codon.In an addition CRF1h is encoded by a cryptic exon immediately after exon 4 (Pisarchik & Slominski, 2001;Slominski et al., 2006b).Similarly to artificial constructs -mNT-CRFR1 (Perrin et al., 2001) and sCRF2α (Chen et al., 2005) the soluble isoform CRF1h may be capable of binding the ligands.This is in contrast to CRF1e isoform missing most of its eCD (Figs. 2 and  3) (Slominski et al., 2006b).Although activity of different artificial models of eCD was proven, their ability to bind substrates was found to be lower when compared to full length receptor.For instance, rat rCRFR1-NT-Kif construct (rat model of eCD), specifically bound to arrestin and rat UCN, but the affinity was found to be 10-folds lower than for full-length rat CRF1 (Hofmann et al., 2001).Similarly, binding of several ligands (rUCN-1, mUCN-2, mUCN-3, r/hCRF) to the eCD1-CRFR2β construct (containing eCD of CRF2β receptor) was 5 to 10 times lower when compared to full length CRF2β expressed in Chinese hamster ovary cell line (CHO).The same study revealed that eCD1-CRFR2β soluble receptor was not able to bind sauvagine, suggesting that another part of CRF2 receptor was specifically responsible for sauvagine binding (Perrin et al., 2003).Other experiments showed involvement of extracellular coil eC-3 in ligand binding and this finding might partially explain lower substrate affinity of soluble CRF receptor isoforms containing exclusively eCD (Sydow et al., 1997;Gkountelias et al., 2009).
Overexpression of CRF1h in immortalized human HaCaT keratinocytes results in co-localization with endoplasmic reticulum and cytoplasm, but not with cell membrane or CRF1α (Zmijewski & Slominski, 2009a).Interestingly, both CRF1h and to a lower degree CRF1e (Zmijewski & Slominski, 2009a), similarly to mNT-CRFR1 (Perrin et al., 2001) and sCRF2α (Chen et al., 2005), could be released to the medium.Thus, localization of soluble isoforms might determine their function.Co-expression of CRF1h with CRF1α increased in production of cAMP in COS-7 cells (Pisarchik & Slominski, 2004), but caused decrease or had no influence on the activity of cAMP or AP-1 responsive elements in Ha-CaT keratinocytes (Zmijewski & Slominski, 2009a) and AtT-20 pituitary cells (Zmijewski & Slominski, 2009b), suggesting involvement of other CRF1 isoforms or/and cell specific factors.Potential activity of secreted soluble isoforms was shown in a "media exchange" experiment performed on AtT-20 cells overexpressing CRF1 isoforms.Presence of CRF1h and to a lesser extend CRF1e in culture medium partially inhibited stimulation of CRF1α by CRF in AtT-20 corticotrophic cells (Zmijewski & Slominski, 2009b).Thus these isoforms, under certain conditions, could act as soluble decoy receptors, as previously suggested (Pisarchik & Slominski, 2004;Chen et al., 2005).
CRF1e protein has only 40 amino acids of native protein including a signal peptide of 23 amino acids.The major part of eCD responsible for ligand binding is missing and the remaining 104 amino acids incorporated to the sequence due to frameshift have no homology to any known protein (Pisarchik & Slominski, 2001).In addition, it seems that CRF1e mRNA, or/and protein is not stable because GFP signal from fusion receptor CRF1e-GFP fades away a few days after transfection, while overexpression of other fusion constructs containing CRF1 isoforms resulted in stable fluorescence for more than a week (Zmijewski & Slominski, 2009a).CRF1e was equally distributed within the cells when overexpressed in HaCaT keratinocytes or AtT-20 pituitary cells.In addition, CRF1e was the sole receptor isoform found in the nucleus.Co-expression of CRF1e and CRF1α did not result in co-localization of both isoforms, but unexpected intracellular aggregation of CRF1α was observed (Zmijewski & Slominski, 2009a).Further studies are necessary to establish a mechanism for those phenomena.Transient transfection with CRF1e did not result in activation of CRF-driven signaling in cells of various origins (Pisarchik & Slominski, 2004;Zmijewski & Slominski, 2009a;2009b).It is possible that accumulation of CRF1e mRNA can lower the pull of CRF1α mRNA spliced out from common precursor, having a negative effect on final CRF1α expression.This hypothesis requires additional experimental validation.

ROle OF AlTeRNATIVe sPlICING
It has been widely accepted that the expression of certain types of GPCR receptors does not fully explain the variety of cellular responses after stimulation with selective agonists.Many factors could be involved in such regulation, including posttranslational modification, receptor oligomerization, constitutive activation or presence of cell specific modifiers (Nelson & Challiss, 2007;Zmijewski & Slominski, 2009a).
Alternative splicing is a common feature of at least 70 % of human genes being responsible for increased functional capacity of the human genome.Many GP-CRs including CRF1 and other members of the B1 family (secretins) have several variants generated by alternative splicing (einstein et al., 2008).Although the detailed mechanism of alternative splicing has been studied for many years, its function is not well understood, especially in the case of GPCRs (Minneman, 2001;Bjarnadottir et al., 2007).
expression of the majority of alternative spliced variants of CRF1 results in a dominant negative phenotype, or at least modulatory responses (Fig. 5).Moreover, alternative splicing of CRF1 is not only tissue-or organ-specific, but it is also tightly regulated by intra and extracellular factors (Hillhouse & Grammatopoulos, 2006;Slominski et al., 2006b;Zmijewski & Slominski, 2009a).In addition, alternatively spliced mRNA of CRF1 isoforms may undergo fast mRNA decay process (nonsense-mediated decay -NMD).This process may be linked to the presence of premature termination codon introduced by alternative splicing (Lewis et al., 2003;Amrani et al., 2006).Several CFR1 isoforms including CRF1e, f, h and k, possess alternative, premature stop codons introduced by alternative splicing (Fig. 2).It seems unlikely that such a complex system, conserved in evolution is just a random noise of the spliceosome.
There is growing evidence that alternative splicing of CRF1 pre-mRNA is responsible for modulation of CRF signaling.In the current model, isoform CRF1α has a dominant role as a fully functional receptor and the remaining isoforms regulate its activity (Fig. 5).The first and obvious consequence of alternative splicing of CRF1 mRNA is lowering of the pool of mRNA of native receptor (CRF1α), which may potentially inhibit of CRF signaling.Another function of CRF1 isoforms may be formation of receptor oligomers (dimers) (Sirianni et al., 2005;Gurevich & Gurevich, 2008).Although dimerization does not appear to be essential for ligand binding (Kraetke et al., 2005), CRF1 was found to form homo-and heterooligomers by several groups (Kraetke et al., 2005;Mikhailova et al., 2007;Young et al., 2007;Zmijewski & Slominski, 2009a).High molecular complexes containing CRF1 isoforms were stable under mild denaturating conditions during SDS/PAGe separation (Sydow et al., 1997;Pisarchik & Slominski, 2004;Slominski et al., 2007b;Zmijewski et al., 2007;Zmijewski & Slominski, 2009a).Interestingly, such complexes are sensitive to deglycosylation and reducing agents (dithiothreitol) suggesting a role of N-glycans and disulfide bonds in the stability of CRF1 ternary complexes (Zmijewski & Slominski, 2009a).The formation of homo-and heterodimers of CRF1 isoforms might also regulate of CRF1α localization and activity.For example, heterooligomerization could explain intracellular co-localization of CRF1α with isoforms with impaired 7TM domains (CRF1d, f and g) and resulting partial inhibition of CRF signaling.Recently a similar interaction was observed for two isoforms of CRF2, e.g., CRF2β, and (iv)-mCRF2β (Sztainberg et al., 2009).Moreover, CRF1β was found to be rapidly internalized after formation of heterodimers with CRF1α.Therefore, we postulated that heterooligomerization of CRF1 isoforms can diversify the repertoire of functional CRF1 receptors and/or modulate CRF signaling (Slominski et al., 2006b;Markovic et al., 2008;Zmijewski & Slominski, 2009a;2009b).Soluble isoforms of CRF1 (CRF1e, h) are similar to mNT-CRFR1 (Perrin et al., 2001) and sCRF2α (Chen et al., 2005) and their biological activities have already been demonstrated.The mechanism of extracellular activity of soluble isoforms should be similar to CRF binding protein (CRF-BP), which serves as an extracellular binding protein that resembles decoy receptors (Chen et al., 2005).
It must be also mentioned that the activity of the receptors from class B of GPCR can be modulated by several other factors, including receptor activity-modifying proteins (RAMPs) (Sexton et al., 2006), β-arrestins (Holmes et al., 2006), G protein-coupled receptor kinases (GRK1-3) or Rab GTPases (Miller et al., 2007).

CRF1 IN HuMAN dIseAses, AlTeRNATIVe sPlICING As A POssIble TARGeT
GPCRs are a target for 40-50 % of all marketed drugs (Hemley et al., 2007) and CRF1 antagonists and agonists have potential benefits in the therapy of several human pathologies.The agonists of CRF1 receptor with low hypotensive activity such as [D-Glu20]-CRH may be used in therapy of malignant melanoma as their antiproliferative activity was shown in vivo and in an animal model (Carlson et al., 2001).Human and rodent skin cancer cell lines including melanomas (Pisarchik & Slominski, 2001;2002), squamous cell carcinomas and basal cell carcinomas (Pisarchik & Slominski, 2001) express different patterns of CRF1 isoforms, thus alternative splicing of CRF1 may play an important role in sensitivity of cancer cells to CRF and its analogues.In general skin cancer cell lines express multiple isoforms of CRF1 receptor.It has to be stressed that the effect of CRF and CRF-related peptides strongly depends on cellular context as has been shown for different skin cells (Zbytek et al., 2003;2004;2005;2006a;2006b;Slominski et al., 1999;2000c;2006b;2007a).
Single nucleotide polymorphism (SNP) another factor, which might also influence alternative splicing of CRF1 mRNA.This phenomenon is of particular interest, because CRF1 SNPs have been associated with many human conditions, including: depression (Liu et al., 2006), stress, alcohol abuse (Blomeyer et al., 2008;Schmid et al., 2009), suicidal tendencies (Wasserman et al., 2008), infection susceptibility, asthma (Lima et al., 2009) and bone density (Jones et al., 2008).Thus, in-depth investigation of CFR1 splicing in the context of CRF1 SNPs might bring some mechanistic explanation for genetically determined changes in CRF1 receptor activity.
Certaily, detailed studies on CRF1 alternative splicing and possible influence of local environment are required in order to assess potential benefits of CRF1 agonist/ antagonist therapy (Wei & Slominski, 2002;Vale, 2004;Pisarchik & Slominski, 2006).On the other hand modulation of CRF1 activity by an alternative splicing may represent a promising strategy to regulate sensitivity of cells to CRF1 agonists or antagonists.There are several factors and drugs with proven influence on alternative splicing (for review see: Sumanasekera et al., 2008).Surprisingly, many anticancer drugs including daunorubicin and cisplatin can also affect alternative splicing as shown for Bcl-X and other human apoptotic genes (Shkreta et al., 2008).Thus, selective modulation of CRF1 splicing is feasible.For example, splice-switching oligonucleotides (for recent review see: Bauman et al., 2009) or chemically modified antisense oligonucleotides (Kole et al., 2004) could be used to change the pattern of expression of CRF1 isoforms in order to sensitize or desensitize cells to CRF1 agonists.Specifically, induction of CRF1 variants c, d, f or g by CRF1-targeted splice-switching oligonucleotides might be alternative to antalarmin in treatment of anxiety disorders, alcohol and drug addiction, inflammatory bowel syndrome.On the other hand, it is possible that directing alternative splicing towards expression of the CRF1α isoform may sensitize cells to CRF-driven inhibition of cancer cell proliferation (for example, melanoma).Thus, induced modulation of alternative splicing of CRF1 may represent a dawn of new therapeutic strategies targeting a variety of diseases (Pisarchik & Slominski, 2006).

ConClusion
There is growing evidence that alternative splicing of CRF1 receptor represents an additional level of regulation of central and local responses to stress via the HPA axis.expression of several CRF1 isoforms depends on a variety of external and internal factors and appears to be responsible for regulation of activity of CRF signaling.Thus, the alternative splicing of CRF1 (and other GPCRs) is not only a very important and complex regulatory mechanism but may also represent a novel pharmacological target in therapy of various disorders (Wei & Slominski, 2002;Hillhouse & Grammatopoulos, 2006;Pisarchik & Slominski, 2006;Slominski et al., 2006b;einstein et al., 2008;Wasserman et al., 2008;Lima et al., 2009;Sztainberg et al., 2009;Zmijewski & Slominski, 2009a).

Figure 1 .
Figure 1.Mechanism of systemic response to stress through activation of the hypothalamic-pituitary-adrenal axis (HPA).The signaling cascade is initiated by corticotropin releasing factor production (CRF) in the hypothalamus, followed by activation of the CRF receptor (CRF1) in the pituitary and subsequent synthesis of pro-opiomelanocortin (POMC).POMC-derived peptides: adrenocorticotropic hormone (ACTH), melanocortin (MSH) and endorphin (END), activate a series of key metabolic pathways including steroidogenesis in the adrenal gland (ACTH) and melanogenesis in the skin (MSH, ACTH and END).Synthesis and release of cortisol inhibits the immune response and form a negative feedback loop for CRF and POMC synthesis.In addition ACTH can trigger melanogenesis in the skin, and MSH, melanin and intermediates of melanogenesis suppress immune response.Modified after(Slominski et al., 2000b).See text and citations within for details.

Figure 2 .
Figure 2. schematic diagram of alternatively spliced isoforms of CRF1 CRF1 splicing variants differ in the number of exons (filed squares).Removed exons were shown as a line and exons with frame-shift as empty squares.Number of the exon (1 to 14) is indicated on the top.Names of CRF1 isoforms are indicated on the left.Isoforms α, β, c, d, e, f, g and h were detected in humans, mouse or hamster.CRF1e2 and CRF1h2 are theoretical alternative transcript from the same mRNA of CRF1e and h, respectively.Isoforms j, k, m and n were found only in hamster and isoform oCRF1var in sheep.Greek letters and doted boxes indicate transmembrane fragments I to VII.Modified after(Slominski et al., 2006b).

Figure 3 .
Figure 3. Model of CRF1 receptor and its splicing variants (α, β, c-h) TM I-VII: transmembrane α-helical fragments, ECD: extracellular domain (ligand binding domain), EC I-III: extracellular coils, IC I-III: intracellular coils.Fragments removed by alternative splicing are shown by dotted lines.Fragments of ECD and EC III taking part in ligand binding are shown in yellow.Fragment of IC III responsible for G-protein binding shown in blue.Thick red dotted line: disulfide bonds, pink structures: glycosylation sides, orange balls: phosphorylation sides.

Figure 4 .
Figure 4. Activation of intracellular signaling pathways by stimulation of CRF1 receptorLigand binding to CRF1 receptor activates at least three G α subunits (α s , α i and α q ) of G-protein resulting in stimulation cAMP production by adenylate cyclase (AC) with subsequent cAMP-induced phosphorylation of CREB and activation of the cAMP-responsive element (CRE).Mobilization of calcium (Ca +2 ) results in activation of the calcium-responsive element (CARE).Activation of AP-1 can also trigger the mitogen-activated protein kinase (MAPK) pathway through protein kinase A (PKA).In addition, α q -mediated stimulation of phospholipase C results in IP 3 -driven activation of activator protein 1 (AP-1) dependent promoters.Downstream signaling from CRF1 receptor regulates expression of several genes including POMC, several interleukins, involucrine and cytokeratin 14.The phenotypic effects of stimulation include: stimulation of differentiation, steroidogenesis, melanogenesis and release of arachidonic acid.CRF inhibits cell division and regulates immune response.See text and citations within for details.

Figure 5 .
Figure 5. Regulation of CRF signaling by CRF1 isoforms CRF1 gene contains 14 exons and only one isoform of receptor-CRF1β (also called pro-CRF1) is coded by all exons.CRF1 transcript is also subjected to alternative splicing resulting in at least 8 isoforms.Recent studies showed that expression and/or co-expression of CRF1 isoforms is responsible for modulation of CRF1 signaling.Plus indicates stimulation of downsteam signaling by the classic pathway (CRF1α) or alternative pathway (CRF1β).Soluble isoforms (CRF1e and h) were also found to stimulate CRF signaling when co-expressed with CRF1α.Minus indicates inhibition of CRF signaling on different levels including: fast mRNA decay (CRF1e), dimerization and subsequent intercellular retention resulting in most probable premature receptor degradation (CRF1α with CRF1d, CRFf or CRFg), decoy receptor mechanism (CRF1h and e when secreted), agonist binding impairment (CRF1c) or G-protein wbinding inhibition (CRF1d).See text for details and citations.