Abstract
Prokineticins are a pair of signal factors involved in many physiological processes by binding to two closely related G-protein-coupled receptors, PKR1 and PKR2. Recently, mutations in prokineticin 2 (PK2) and PKR2 are found to be associated with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, disorders characterized by delayed puberty and infertility. However, little is known how PKRs interact and activate G-proteins to elicit signal transduction. In the present study, we took advantage of one disease-associated mutation (R164Q) located in the second intracellular (IL2) loop of PKR2, to investigate the role of IL2 loop in the cell signaling, G-protein binding and receptor trafficking. R164Q mutant PKR2 showed normal cell surface expression and ligand binding capacity. However, the PKR2 signaling was abolished by R164Q mutation. We demonstrated that R164Q mutation disrupted the interaction of IL2 loop to the Gα(q), Gα(i), and Gα(16)-proteins. A positive-charged amino acid at this position is required for proper function, and the signaling efficacy and potency depend on the net amount of positive charges. We also demonstrated that the interactive partner of Arg-164 may localize in the C-terminal five residues of Gα(q)-protein. A series of mutation analysis indicated that the basic amino acids at the C terminus of IL2 loop may function cooperatively in GPCRs. Furthermore, R164Q mutation also results in minimal ligand-induced endocytosis of PKR2. As many GPCRs share structural homology in the C terminus of IL2 loop, our findings may have general application in understanding structure and function of GPCRs.
Highlights
Ture of G-protein-coupled receptors (GPCRs) consists of seven transmembrane (TM) helices, with three intracellular loops (IL1–3) and the cytoplasmic C-terminal tail
Genetic studies have shown that mutations in prokineticin 2 (PK2) and PKR2 are associated with the Kallmann syndrome (KS) and/or idiopathic hypogonadotropic hypogonadism (IHH), disorders characterized by delayed puberty and infertility (9 –13)
Because previous work has indicated R164Q-PKR2 showed intact ligand binding capacity (16), it is very likely that R164Q substitution affects G-protein coupling
Summary
Constructs—The cDNAs for human PKR1 and PKR2 was kindly provided by Dr Qun-Yong Zhou (University of California, Irvine). Chinese Hamster Ovary (CHO) cells stably expressing the photoprotein aequorin were transiently transfected with wild-type PKR1/PKR2 or their respective mutants. PK2 (a kind gift from Dr Qun-Yong Zhou, University of California at Irvine) were serially diluted in Hank’s balanced salt solution plus 10 mM HEPES, pH 7.5, and 0.1% bovine serum albumin. After washing with buffer A, the beads were resuspended in 1 ml of buffer A containing lysates from cells transiently expressed with Flag-G␣16, HA-G␣q, and HA-G␣qi, respectively. Stable transfected HEK293 cells were subcultured onto poly-D-lysine-coated glass slides in 12-well dishes and incubated with cycloheximide (Sigma) for 4 h before experiments. The Alexa Fluor 594 was excited at 590 nm and detected with a long pass band filter at 617 nm
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