IDENTIFICATION OF MEMBRANE INSERTION SEQUENCES OF THE RABBIT GASTRIC CHOLECYSTOKININ-A RECEPTOR BY IN-VITRO TRANSLATION

To determine which amino acid sequences account for transmembrane fold ing of G7 receptors, the membrane domain of the rabbit cholecystokinin -A (CCK-A) G-protein-coupled receptor has been investigated by in vitr o transcription/translation of two types of fusion vectors containing sequences that include putative transmembrane segments. First, the sev en putative transmembrane domains of the CCK-A receptor were inserted individually into pGEM vectors beginning with the cDNA encoding the fi rst 101 (HK-M0) or 139 (HK-M1) amino acids of the alpha subunit of the gastric H,K-ATPase. These were separated by the cDNA for the inserted transmembrane domains from the cDNA encoding the last 177 amino acids of the beta subunit of the H,K-ATPase containing five N-linked glycos ylation consensus sequences (Bamberg, K., and Sachs, G. (1994) J. Biol . Chem. 269, 16909-16919). Transcription/translation of these fusion v ectors in rabbit reticulocyte lysate +/- dog pancreatic microsomes fol lowed by SDS-polyacrylamide gel electrophoresis defined the presence o f signal anchor sequences in HK-M0 by glycosylation and stop transfer sequences in HK-M1 by inhibition of glycosylation. Six out of the seve n putative transmembrane domains had membrane insertion signals, but n o membrane insertion activity was found for the H3 segment in these ve ctors. To test the effect of specific upstream and downstream sequence s on membrane insertion, vectors were also made starting with the cDNA encoding the N terminus of the CCK-A receptor separated from the last 177 amino acids of the H,K-ATPase beta subunit by cDNA encoding CCK-A receptor sequences of different lengths. In addition to transcription /translation, endoglycosidase H treatment was used to verify glycosyla tion when multiple bands were found in the presence of microsomes. The four positive charges in the loop between H1 and H2 were required for the correct orientation of the first transmembrane domain. The H3 seg ment acted as a stop transfer sequence only when the whole N terminus and H3 were followed by the positive charges in the cytoplasmic loop b etween H3 and H4. The activity of H6 as a signal anchor sequence depen ded on preceding positive charges. These translation data using two ty pes of fusion vectors establish a seven-transmembrane folding model us ing only in vitro translation for the CCK-A receptor beginning with tw o signal anchor sequences and then alternating stop transfer and signa l anchor insertions. Positive charges between H1 and H2, H3 and H4, an d H5 and H6 function as cytoplasmic anchors in the membrane folding of this receptor.