The cGMP kinase signaling complex identified previously in tracheal smooth muscle membranes contains a number of cGMP kinase substrates termed GO through G4, GO G1, and G2 were identified as IP3 receptor I (IP3RI). IRAG, and cGMP kinase I. Sequencing of purified G3 and G4 showed that these proteins were proteolytic cleavage products of IRAG. However. the purified cGMP kinase signaling complex contained following additional proteins: alpha-actin, calponin H1. and phospholamban (PLB) as verified by MALDI-TOF as well as MS/MS sequencing and immune detection. The complex of these six proteins was immune precipitated by, antibodies to each protein. The proteins were phosphorylated by the endogenous cGMP kinase I with the exception of a-actin and calponin H1. The complex did not contain the Ca2+-ATPase SERCA H. PLB. IP3RI. and cGMP kinase Ibeta were coimmune precipitated after expression in COS-7 cells. These results Suggest that PLB may have additional functions to regulate the activity of SERCA II. (C) 2002 Elsevier Science (USA). All rights reserved.