CONTROL OF SURFACTANT SECRETION BY GTP BINDING PROTEINS

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Alveolar type 2 pneumocytes synthesize and secrete lung surfactant in both constitutive and regulated modes. We have obtained evidence that GTP binding proteins participate in this pathway at two levels. First, several species of transductional G-proteins which couple surface receptors to effector molecules responsible for generating second messengers are present in type 2 cell membranes and cytosol. The presence of Gs, which couples the B- adrenergic receptor to adenylyl cyclase, had been implied by the earlier studies of others. We have obtained direct evidence for the presence of two species of Gi in addition to Gs. Further, we have shown that direct stimulation of the Gi proteins causes rapid and dramatic surfactant secretion. Preliminary evidence for the basis of stimulus-response coupling by this pathway is presented. Second, we have identified and partially purified three low molecular weight ras-related GTP binding proteins from lamellar bodies. Recent evidence from yeast genetics and mammalian in vitro complementation studies suggests that members of this family play key roles in the sorting and exocytosis of secretory vesicles. The goals of this project are to identify and characterize at a physiologic and molecular level the GTP binding proteins involved in stimulus-secretion coupling in type 2 cells. These goals will be pursued through three specific aims. 1) We will identify and quantitate the transductional G-protein species present in type 2 cells, establish the role of Gi proteins in mediating surfactant secretion and characterize the second messenger systems involved, and examine receptor mediated activation of this pathway. 2) We will purify low molecular weight GTP binding proteins from lamellar bodies, obtain partial protein sequence, clone cDNAs, produce anti- peptide antisera, and characterize the subcellular distribution and physiologic roles of these proteins. We will also determine if other low molecular weight GTP-binding proteins are present in non- lamellar body compartments of the type 2 cell. 3) Functional coupling of GTP binding proteins will be examined in cultured type 2 cells as they lose, or are induced by matrix to regain, their surfactant secretory function, and in type 2 cells derived from fetal lungs which can be induced to express features of differen- tiation in vitro. Completion of these studies should expand our knowledge of the molecular mechanisms regulating surfactant secretion by type 2 cells and will provide tools for studying maturational events in the alveolar epithelium.