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Molecular and functional characterization of novel CRFR1 isoforms from the skin
Alexander Pisarchik1, Andrzej Slominski
1Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Abstract:
In our continued studies on corticotropin releasing factor receptor (CRFR1) signaling in the skin, we tested functional activity of CRFR1alpha, e, f, g and h isoforms after transfection to COS cells. Both membrane-bound and soluble variants are translated in vivo into final protein products that undergo further post-translational modifications. CRFR1alpha was the only isoform coupled directly to adenylate cyclase with the exception of an artificial isoform (CRFR1h2) with the insertion of 37 amino acids between the ligand binding domain and the first extracellular loop that was capable of producing detectable levels of cyclic AMP (cAMP). Soluble isoforms could modulate cell response with CRFR1e attenuating and CRFR1h amplifying CRFR1alpha-coupled cAMP production stimulated by urocortin. Testing with plasmids containing the luciferase reporter gene, and inducible cis-elements (CRE, CaRE, SRE, AP1 or NF-kappaB) demonstrated that only CRFR1alpha was involved directly in the transcriptional regulation, while CRFR1g inhibited CRE activity. Significantly higher reporter gene expression by CRF was observed than that mediated by 4beta-phorbol 12-myristate 13-acetate and forskolin alone, being compatible with the concomitant treatment by phorbol 12-myristate 13-acetate and forskolin. This suggests that both protein kinase A and C can be involved in CRF-dependent signal transduction.
Insights
Corticotropin releasing factor receptor (CRFR1) isoforms exhibit distinct signaling functions. CRFR1alpha directly activates adenylate cyclase, while other isoforms modulate this response, impacting skin cell signaling pathways.
Area of Science:
- Dermatology
- Molecular Biology
- Endocrinology
Background:
- Corticotropin releasing factor receptor (CRFR1) signaling plays a role in skin physiology.
- Multiple CRFR1 isoforms (alpha, e, f, g, h) exist, with varying functional activities.
- Understanding these isoforms is crucial for elucidating CRFR1-mediated cellular responses in the skin.
Purpose of the Study:
- To investigate the functional activity of different CRFR1 isoforms (CRFR1alpha, e, f, g, h).
- To determine the specific signaling pathways and transcriptional regulation mediated by these CRFR1 isoforms.
- To explore the modulatory effects of soluble CRFR1 isoforms on CRFR1alpha-coupled signaling.
Main Methods:
- Transfection of COS cells with plasmids encoding CRFR1 isoforms.
- Measurement of cyclic AMP (cAMP) production to assess adenylate cyclase coupling.
- Luciferase reporter gene assays to evaluate transcriptional regulation via cis-elements (CRE, CaRE, SRE, AP1, NF-kappaB).
Main Results:
- CRFR1alpha was the sole isoform directly coupled to adenylate cyclase, producing cAMP.
- Soluble isoforms CRFR1e and CRFR1h modulated CRFR1alpha-stimulated cAMP production, with CRFR1e attenuating and CRFR1h amplifying the response.
- CRFR1alpha directly mediated transcriptional regulation, while CRFR1g inhibited CRE activity.
- CRF stimulation resulted in higher reporter gene expression than phorbol esters or forskolin alone, suggesting involvement of protein kinase A and C.
Conclusions:
- CRFR1 isoforms possess distinct signaling capabilities, influencing cellular responses.
- CRFR1alpha plays a key role in cAMP production and transcriptional regulation.
- Soluble CRFR1 isoforms act as modulators of CRFR1alpha signaling.
- CRF-dependent signal transduction involves both protein kinase A and C pathways in the skin.
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