Abstract
We recently identified a novel mechanism explaining how the mouse (m) prostacyclin receptor (IP) couples to Galpha(s), Galpha(i), and Galpha(q) (Lawler, O. A., Miggin, S. M., and Kinsella, B. T. (2001) J. Biol. Chem. 276, 33596-33607) whereby mIP coupling to Galpha(i) and Galpha(q) is dependent on its initial coupling to Galpha(s) and subsequent phosphorylation by cAMP-dependent protein kinase A (PKA) on Ser(357). In the current study, the generality of that mechanism was investigated by examining the G protein coupling specificity of the human (h) IP. The hIP efficiently coupled to Galpha(s)/adenylyl cyclase and to Galpha(q)/phospholipase C activation but failed to couple to Galpha(i). Coupling of the hIP to Galpha(q), or indeed to Galpha(s) or Galpha(i), was unaffected by the PKA or protein kinase C (PKC) inhibitors H-89 and GF 109203X, respectively. Thus, mIP and hIP exhibit essential differences in their coupling to Galpha(i) and in their dependence on PKA in regulating their coupling to Galpha(q). Analysis of their primary sequences revealed that the critical PKA phosphorylation site within the mIP, at Ser(357), is replaced by a PKC site within the hIP, at Ser(328). Conversion of the PKC site of the hIP to a PKA site generated hIP(QL325,326RP) that efficiently coupled to Galpha(s) and to Galpha(i) and Galpha(q); coupling of hIP(QL325,326RP) to Galpha(i) but not to Galpha(s) or Galpha(q) was inhibited by H-89. Abolition of the PKC site of the hIP generated hIP(S328A) that efficiently coupled to Galpha(s) and Galpha(q) but failed to couple to Galpha(i). Finally, conversion of the PKA site at Ser(357) within the mIP to a PKC site generated mIP(RP354,355QL) that efficiently coupled to Galpha(s) but not to Galpha(i) or Galpha(q). Collectively, our data highlight critical differences in signaling by the mIP and hIP that are regulated by their differential phosphorylation by PKA and PKC together with contextual sequence differences surrounding those sites.
Highlights
This article has been withdrawn by the authors
As the original autoradiograms and scan images relating to the aforementioned figures are no longer available to investigate the matter, the authors wish to withdraw the article in the interests of maintaining their publication standards, while respecting the highest standards of transparency and reliability of their research and of the JBC
Replica data sets for each of the figures in question that the authors state fully validate the findings and conclusions of the published article are available, and, a revised version of the manuscript with the replica data sets can be obtained by contacting the corresponding author
Summary
This article has been withdrawn by the authors. The authors of the paper have become aware that some features had been duplicated in Figs. 7, C and D, and 8G.
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