Identification of transmembrane domains that regulate spatial arrangements and activity of prokineticin receptor 2 dimers

Abstract

The chemokine prokineticin 2 (PK2) activates its cognate G protein-coupled receptor (GPCR) PKR2 to elicit various downstream signaling pathways involved in diverse biological processes. Many GPCRs undergo dimerization that can modulate a number of functions including membrane delivery and signal transduction. The aim of this study was to elucidate the interface of PKR2 protomers within dimers by analyzing the ability of PKR2 transmembrane (TM) deletion mutants to associate with wild type (WT) PKR2 in yeast using co-immunoprecipitation and mammalian cells using bioluminescence resonance energy transfer. Deletion of TMs 5-7 resulted in a lack of detectable association with WT PKR2, but could associate with a truncated mutant lacking TMs 6-7 (TM1-5). Interestingly, TM1-5 modulated the distance, or organization, between protomers and positively regulated Gαs signaling and surface expression of WT PKR2. We propose that PKR2 protomers form Type II dimers involving TMs 4 and 5, with a role for TM5 in modulation of PKR2 function. We have carefully considered the reviewers' comments and suggestions and extensively edited the manuscript to address them, including addition of new data. In particular, we have included further controls to confirm that BRET-tagged receptors are functional and inclusion of BRET analysis in both HEK and CHO cells. We have also made improvements to the text in order to ensure a clearer understanding of the methodology and results. We now believe that we have fully addressed all of the concerns raised by the Reviewers and, therefore, wish to submit the revised manuscript for your consideration.Specific responses to comments together with descriptions of new data added and changes made in the text and figures, are detailed in the attached "Response to Reviewers." We believe that the revised manuscript is substantially improved, and we thank the reviewers for their positive comments and for aiding with refinement and clarity of this study.Thank you again for your consideration, and we hope that you will now find our work acceptable for publication. Yours sincerely, Aylin Hanyaloglu and Rosella MieleCover Letter Response to ReviewersReviewer #1: There is one concern: the backgrounds of the images in figure 2 are different--which suggests either differential processing or alteration of the contrast.We would like to thank the reviewer for their overall positive comments. In regards to Figure 2 we would like to assure the reviewer there was no post-acquisition modification of the figures. The development of film in the laboratory in Rome is carried out without an automated film processor but incubation in individual baths of developer, which can result in background differences of the films. The original films were presented as photographed images in the original submission thus we have now scanned the original films and presented them in grey-scale to improve image clarity (please see new Figure 2) but again we would like to emphasize that no alterations of the contrast were made. We hope th...

Paradoxical gain‐of‐function mutant of the G‐protein‐coupled receptor PROKR2 promotes early puberty

“…However, as Ca 2+ flux curves of the co-transfected cells paralleled that of the wild-type-transfected cells, enhanced signal transduction rather than delayed desensitization appears to be the major effect of p.C242fsX305. These results are consistent with those of a previous study by Sposini et al [12]. Our p.C242fsX305 mutant may modify the spatial arrangement of PROKR2 protomers to form hyperactive heterodimers, as in the case of the TM1-5 variant [12].…”

“…. Our p.C242fsX305 mutant may modify the spatial arrangement of PROKR2 protomers to form hyperactive heterodimers, as in the case of the TM1‐5 variant .…”

“…Thus, although Sposini et al . have shown that the TM1‐5 variant dimerizes with the wild‐type protein, the physical interaction between p.C242fsX305 and wild‐type PROKR2 needs to be confirmed by future studies. Second, subcellular localization of the wild‐type and mutant PROKR2 was not examined this study.…”

“…In addition, all known nonsense and frameshift mutations in PROKR2 satisfied the conditions for nonsensemediated mRNA decay (NMD) [11]. Recently, Sposini et al [12] examined the functional property of artificially created PROKR2 variants. The authors found that a variant (TM1-5) lacking the last two transmembrane domains and the carboxyl-terminal tail exerts a unique effect on signal transduction; although the mutant retained no signal transduction activity, co-transfection of this mutant markedly increased ligand-induced signal responses of cells expressing wild-type PROKR2.…”

Oligomerization of GPCRs involved in endocrine regulation

“…PROKR2 variants have been further described in patients with idiopathic combined pituitary hormone deficits (CPHD) including gonadotropin deficiency (Raivio et al 2012, Reynaud et al 2012. The PROKR2 also forms constitutive homomers in vivo ( Marsango et al 2011), and specific protomer-protomer interfaces for the oligomer have been suggested ( Sposini et al 2015). Future investigation of the relationship between oligomerization and pathogenic biased signaling modulation at this receptor is of interest.…”