Functional characterization of the central hydrophilic linker region of the urea transporter UT-A1: cAMP activation and snapin binding.
Of the three major protein variants produced by the UT-A gene (UT-A1, UT-A2, and UT-A3) UT-A1 is the largest. It contains UT-A3 as its NH(2)-terminal half and UT-A2 as its COOH-terminal half. When being part of UT-A1, UT-A3 and UT-A2 are joined by a segment, Lp, whose central part, Lc, is not part of UT-A3 or UT-A2 but is present only in UT-A1. Lc contains the phosphorylation sites S486 and S499 that are involved in protein kinase A-dependent activation, as well as the binding site for snapin, a protein involved in soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-mediated vesicle trafficking and fusion to the plasma membrane. We attached Lc to UT-A2 and UT-A3 to test how these phosphorylation sites influenced their urea transport activity. Adding Lc to UT-A2 conferred stimulation by cAMP to the cAMP-unresponsive UT-A2, and adding Lc to UT-A3 did not further enhance its already existing cAMP response. These findings suggest that the responsiveness to vasopressin that is observed with UT-A1 can be introduced into the unresponsive UT-A2 variant through the Lc segment that is unique to UT-A1. In UT-A3, however, the Lc segment plays no significant role in its activation by cAMP. In addition, the Lc segment also gave UT-A2 the ability to bind snapin and, in Xenopus oocytes, to be stimulated in its urea transport activity by snapin and syntaxins 3 and 4, in the same way as UT-A1.
Mistry
Abinash C
AC
Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Mallick
Rickta
R
Klein
Janet D
JD
Sands
Jeff M
JM
Fröhlich
Otto
O
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005260:Female; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D009865:Oocytes; D010766:Phosphorylation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014508:Urea / Q000378:metabolism*; D014667:Vasopressins / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis; D000096982:Urea Transporters
D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D014508:Urea; D014667:Vasopressins; D033921:Vesicular Transport Proteins; D000096982:Urea Transporters
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