Specific Inhibition of Glycosylation by Tunicamycin Affects Endothelin Receptors in Cultured Swiss 3T3 Fibroblasts

Abstract

The importance of glycosylation to endothelin (ET) receptors was studied in cultured Swiss 3T3 fibroblasts. ET stimulated phosphatidylinositol hydrolysis by 270% in control cells. This stimulatory effect was decreased to < 130% in tunicamycin (TU)-treated cells. TU inhibited protein glycosylation, but did not effect DNA synthesis, cell morphology, or cell numbers. ET-1 binding was greatly reduced in TU-treated cells. In membranes prepared from control cells, [¹²⁵]ET-1 binding was saturable, and appeared as a straight line in Scatchard analysis with values of 0.7 pmol/mg and 0.9 nM for B_max and K_D, respectively. [¹²⁵]ET-1 binding to membranes from TU-treated cells was not saturable. Scatchard analysis showed a biphasic line. Values of B_max, and K_D for the higher affinity site were 0.17 pmol/mg and 0.12 nM, respectively. The low affinity site exhibited a much weaker affinity for ET with an estimated K_D value of 6.8 nM. Cross-linking studies revealed that the receptor in control membranes was a protein with a molecular mass of 62 kDa. In membranes from TU-treated cells, two proteins with molecular masses of 62 and 40 kDa were labeled. These data suggest that receptor glycosylation was required for ET binding and the subsequent activation of the phosphatidylinositol hydrolysis pathway.

Key Words:

• endothelin
• receptor
• glycosylation
• tunicamycin