The 5-hydroxytryptamine(4a) receptor is palmitoylated at two different sites, and acylation is critically involved in regulation of receptor constitutive activity.
We have reported recently that the mouse 5-hydroxytryptamine(4a) (5-HT(4(a))) receptor undergoes dynamic palmitoylation (Ponimaskin, E. G., Schmidt, M. F., Heine, M., Bickmeyer, U., and Richter, D. W. (2001) Biochem. J. 353, 627-663). In the present study, conserved cysteine residues 328/329 in the carboxyl terminus of the 5-HT(4(a)) receptor were identified as potential acylation sites. In contrast to other palmitoylated G-protein-coupled receptors, the additional cysteine residue 386 positioned close to the COOH-terminal end of the receptor was also found to be palmitoylated. Using pulse and pulse-chase labeling techniques, we demonstrated that palmitoylation of individual cysteines is a reversible process and that agonist stimulation of the 5-HT(4(a)) receptor independently increases the rate of palmitate turnover for both acylation sites. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT(4(a)) receptors were indistinguishable from the wild type in their ability to interact with G(s), to stimulate the adenylyl cyclase activity and to activate cyclic nucleotide-sensitive cation channels after agonist stimulation. The most distinctive finding of the present study was the ability of palmitoylation to modulate the agonist-independent constitutive 5-HT(4(a)) receptor activity. We demonstrated that mutation of the proximal palmitoylation site (Cys(328) --> Ser/Cys(329) --> Ser) significantly increases the capacity of receptors to convert from the inactive (R) to the active (R*) form in the absence of agonist. In contrast, the rate of isomerization from R to R* for the Cys(386) --> Ser as well as for the triple, non-palmitoylated mutant (Cys(328) --> Ser/Cys(329) --> Ser/Cys(386) -->Ser) was similar to that obtained for the wild type.
Ponimaskin
Evgeni G
EG
Abteilung Neuro- und Sinnesphysiologie, Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany.
Heine
Martin
M
Joubert
Lara
L
Sebben
Michele
M
Bickmeyer
Ulf
U
Richter
Diethelm W
DW
Dumuis
Aline
A
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007150:Immunohistochemistry; D007313:Insecta; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019308:Palmitic Acid / Q000378:metabolism*; D010169:Palmitic Acids / Q000737:chemistry; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011985:Receptors, Serotonin / Q000378:metabolism*; D044422:Receptors, Serotonin, 5-HT4; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D010169:Palmitic Acids; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D044422:Receptors, Serotonin, 5-HT4; D019308:Palmitic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012694:Serine; D004247:DNA; D000242:Cyclic AMP; D003545:Cysteine