Abstract
gamma-Aminobutyric acid type C (GABAC) receptors identified in retina appear to be composed of GABA rho subunits. The purpose of this study was to localize signals for homooligomeric assembly of rho1 subunits and to investigate whether the same region contained signals for heterooligomeric interaction with rho2 subunits. In vitro translated human rho1 was shown to be membrane-associated, and proteinase K susceptibility studies indicated that the N terminus was oriented in the lumen of ER-derived microsomal vesicles. This orientation suggested the involvement of the N terminus of rho1 in the initial steps of subunit assembly. To test this hypothesis, mutants were created containing only N-terminal sequences (N-rho1) or C-terminal sequences (C-rho1) of rho1. Co-immunoprecipitation studies revealed that N-rho1, but not C-rho1, interacted with rho1 in vitro. When coexpressed in Xenopus oocytes, N-rho1 interfered with rho1 receptor formation. Together, these data suggested that signals for rho1 homooligomeric assembly reside in the N-terminal half of the subunit. Sequential immunoprecipitations were then performed upon cotranslated rho1 and rho2 subunits which demonstrated that rho1 and rho2 interacted in vitro. Co-immunoprecipitation indicated that N-rho1 specifically associated with rho2. Therefore, the N-terminal regions of rho subunits contain the initial signals for both homooligomeric and heterooligomeric assembly into receptors with GABAC properties.
Highlights
Lung surfactant protein D (SP-D)1 belongs to a group of C-type lectins called collectins [1, 2]
Five collectins have been described, mannan-binding lectin (MBL), conglutinin and collectin-43 (CL-43), which are serum proteins produced by the liver; and lung surfactant proteins A and D (SP-A and SP-D), which have until recently been described as surfactant-associated proteins synthesized by alveolar type II cells and by nonciliated bronchial epithelial cells but both have been found to be present in cells lining the gastrointestinal tract [3, 4]
During the purification of human SP-D from the 10,000 ϫ g supernatant from Bronchioalveolar Lavage (BAL) of a patient suffering from alveolar proteinosis, a molecule with an apparent molecular mass of 340 kDa was observed by SDS-polyacrylamide gel electrophoresis (PAGE) in reducing conditions
Summary
Lung surfactant protein D (SP-D) belongs to a group of C-type lectins called collectins [1, 2]. One was found to bind MBL, conglutinin, CL-43, and SP-A, and this receptor is referred to as the collectin receptor (20 –22) This molecule, which has a high degree of homology with calreticulin, has been observed on many cell types, including macrophages and alveolar type II cells. The other receptor is referred to as the C1q receptor with a molecular mass of 126 kDa. A monoclonal antibody that recognizes this cell surface molecule inhibited the MBL-mediated enhancement of phagocytosis [23]. A monoclonal antibody that recognizes this cell surface molecule inhibited the MBL-mediated enhancement of phagocytosis [23] Neither of these receptors has been shown to bind SP-D. Glycoprotein-340 Is a SP-D Binding Molecule vage from a patient with alveolar proteinosis, and we provide data consistent with this molecule being a secreted form of a putative receptor for SP-D
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