Expression and functional characterization of membrane-integrated mammalian corticotropin releasing factor receptors 1 and 2 in Escherichia coli.
Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2β as well as fragments lacking the N-terminal extracellular domain, were overproduced in E. coli. The characteristics of different CRFR2β-PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2β expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of E. coli bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2β product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2β were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2β membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our E. coli expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.
Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, California, USA.
Roberto
R
Jappelli
Laboratory of Neuronal Structure and Function, Salk Institute for Biological Studies, La Jolla, California, USA.
Marilyn H
MH
Perrin
Laboratory of Neuronal Structure and Function, Salk Institute for Biological Studies, La Jolla, California, USA.
Kathy A
KA
Lewis
Laboratory of Neuronal Structure and Function, Salk Institute for Biological Studies, La Jolla, California, USA.
Joan M
JM
Vaughan
Laboratory of Physical Chemistry, ETH, Zürich, Switzerland.
Christos
C
Tzitzilonis
Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, La Jolla, California, USA.
Jean E
JE
Rivier
Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, La Jolla, California, USA.
Wylie W
WW
Vale
Laboratory of Physical Chemistry, ETH, Zürich, Switzerland.
Roland
R
Riek
D029845:Amphibian Proteins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008322:Mammals / Q000378:metabolism*; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism; D036361:Peptide Hormones / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D000099052:CRF Receptor, Type 1
D029845:Amphibian Proteins; C101531:CRF receptor type 2; D003470:Culture Media; D003902:Detergents; D008024:Ligands; D010446:Peptide Fragments; D036361:Peptide Hormones; D021382:Protein Sorting Signals; D018019:Receptors, Corticotropin-Releasing Hormone; D011993:Recombinant Fusion Proteins; C104416:astressin; D000099052:CRF Receptor, Type 1; C025727:sauvagine; D003346:Corticotropin-Releasing Hormone