Functional characterization and kinetic studies of an ancestral lamprey GnRH-III selective type II GnRH receptor from the sea lamprey, Petromyzon marinus.
The recently cloned lamprey GnRH receptor was shown to have several unique features, including the longest intracellular C-terminal tail (120 amino acids (aa)) of any previously described GnRH receptor. In the current study, a series of experiments were performed examining cAMP responses, binding kinetics, whole cell competitive binding assays and internalization studies of the lamprey GnRH receptor using a series of three C-terminal tail truncations (80 aa, 40 aa and 0 aa) to better describe the functional significance of this unique vertebrate GnRH receptor. Activation of the lamprey GnRH receptor was shown to stimulate cAMP production in a dose-dependent manner when treated with either lamprey GnRH-I (LogEC50 -6.57+/-0.15) or lamprey GnRH-III (LogEC(50) -8.29+/-0.09). Truncation analysis indicated that the membrane proximal 40 aa of the lamprey GnRH receptor C-terminal tail contain a motif required for cAMP accumulation. Saturation binding assays using the wild type and truncated lamprey GnRH receptors revealed that all of three truncated lamprey GnRH receptors were capable of binding lamprey GnRH-I. Competitive, intact cell-binding assays suggested that the lamprey GnRH receptor is lamprey GnRH-III selective, based on the observed pharmacological profile: lamprey GnRH-III (Inhibitory constant (Ki) 0.708+/-0.245 nM)=chicken GnRH-II (Ki 0.765+/-0.160 nM) > mammalian GnRH (Ki 12.9+/-1.96 nM) > dAla(6)Pro(9)NEt mammalian GnRH (Ki 21.6+/-9.68 nM) > lamprey GnRH-I (Ki 118.0+/-23.6). Finally, the lamprey GnRH receptor was shown to undergo rapid ligand-dependent internalization, which was significantly diminished in the tail-less truncated form. We have shown from our current and our previous structural studies that this unique lamprey GnRH receptor shares several characteristics of both type I and type II GnRH receptors which suggests that this receptor has retained ancestral characteristics that can provide insight into the function and evolution of the vertebrate GnRH receptor family.
Silver
M R
MR
Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824, USA.
Sower
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D048428:Petromyzon; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D011966:Receptors, LHRH / Q000235:genetics / Q000378:metabolism*
D009842:Oligopeptides; D020033:Protein Isoforms; D011966:Receptors, LHRH; C448022:gonadotropin-releasing hormone-III; D007987:Gonadotropin-Releasing Hormone; D000242:Cyclic AMP; D011761:Pyrrolidonecarboxylic Acid