SYNTHESIS AND PROCESSING OF GLYCOPHORIN A IN K562 ERYTHROLEUKEMIA CELLS

Abstract

I have studied the homologous desensitization of agonist stimulated adenylate cyclase in adipocytes containing either the (alpha)(,1) or (alpha)(,2) receptor. Adipocytes differentiated with dexamethasone and methylisobutylxanthine (MIX) contain (alpha)(,2) adrenergic receptors coupled to adenylate cyclase that becomes rapidly desensitized upon incubation with isoproterenol. In contrast, adenylate cyclase that couples to (alpha)(,1) adrenergic receptors in adipocytes differentiated using insulin and MIX does not become desensitized.;The inhibitory guanine nucleotide regulatory component (Gi) of adenylate cyclase was assayed in 3T3-L1 cells by Western immunoblot analysis. Gi decreases several fold in 3T3-L1 cells during differentiation. The highest level of Gi was attained in confluent fibroblasts or in non-differentiating 3T3-C2 cells. The lowest level of Gi was observed in cultures of mature adipocytes six days post confluence. This is the first evidence that the Gi protein is subject to developmental regulation.;I have prepared and characterized monoclonal antibodies to glycophorin A and to band III from human erythrocyte membranes.;The glycophorin A monoclonal antibody was used for detailed studies of the biosynthesis and processing of glycophorin A in human K562 erythroleukemia cells. Glycosylation inhibitors and glycosidases were used to determine the kinetics of oligosaccharide processing events in K562 cells. A 27 kDa N-linked glycosylated precursor is synthesized within a 5 minute pulse of ('35)S methionine. The 27 kDa initial precursor is rapidly converted to a 31 kDa intermediate in a proximal region of the Golgi complex by the addition of multiple galNAc residues to Ser/Thr hydroxyl groups. Subsequent maturation within the Golgi complex, converting the 31 kDa polypeptide to 42 kDa, involves the processing of the high mannose chain to a complex type oligosaccharide and the concomitant addition of galactose and sialic acid to galNAc residues of the O-linked oligosaccharide chains. Glycophorin A mRNA isolated from K562 cells programmed the synthesis of a 24 kDa polypeptide product. The 24 kDa polypeptide is also observed when the 27 kDa precursor is treated with endoglycosidase H in vitro. The processing on the N-linked oligosaccharide chain and the formation of the 15 O-linked oligosaccharide chains are dramatically slowed in the presence of the ionophore, monensin, pinpointing these events to the Golgi apparatus. (Abstract shortened with permission of author.).