Carboxyl tail cysteine mutants of the thyrotropin-releasing hormone receptor type 1 exhibit constitutive signaling: role of palmitoylation.
We studied the role of carboxyl tail cysteine residues and their palmitoylation in constitutive signaling by the thyrotropin-releasing hormone (TRH) receptor type 1 (TRH-R1) in transfected mammalian cells and in Xenopus laevis oocytes. To study palmitoylation, we inserted a factor Xa cleavage site within the third extracellular loop of TRH-R1, added a carboxyl-terminal C9 immunotag and expressed the mutant receptor in Chinese hamster ovary cells. We identified TRH-R1-specific palmitoylation in the transmembrane helix-7/carboxyl-tail receptor fragment mainly at Cys-335 and Cys-337. In contrast to a mutant truncated at Cys-335 that was reported previously to be constitutively active, a receptor truncated at Lys-338 (K338Stop), which preserves Cys-335 and Cys-337, and C337Stop and N336Stop, which preserve Cys-335, did not exhibit increased constitutive signaling. TRH-R1 mutants substituted singly by Gly or Ser at Cys-335 or Cys-337 did not exhibit constitutive signaling. By contrast, substitution of both cysteines (C335G/C337G or C335S/C337S) yielded TRH-R1 mutants that exhibited marked constitutive signaling in mammalian cells. In the oocyte, constitutive signaling by C335G/C337G resulted in homologous (of C335G/C337G) and heterologous (of M1 muscarinic receptor) desensitization. Because both Cys-335 and Cys-337 have to be substituted or deleted for constitutive signaling, we propose that a single palmitoylation site in the proximal carboxyl tail is sufficient to constrain TRH-R1 in an inactive conformation.
Du
Dongyi
D
Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-8029, USA.
Raaka
Bruce M
BM
Grimberg
Hagit
H
Lupu-Meiri
Monica
M
Oron
Yoram
Y
Gershengorn
Marvin C
MC
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics*; D005260:Female; D006801:Humans; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics* / Q000502:physiology; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000235:genetics* / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics*; D014982:Xenopus laevis
D010446:Peptide Fragments; D018025:Receptors, Thyrotropin-Releasing Hormone; D019308:Palmitic Acid; D003545:Cysteine