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 folding of G7 receptors, the membrane domain of the rabbit cholecystokinin-A (CCK-A) G-protein-coupled receptor has been investigated by in vitro transcription/translation of two types of fusion vectors containing sequences that include putative transmembrane segments. First, the seven putative transmembrane domains of the CCK-A receptor were inserted individually into pGEM vectors beginning with the cDNA encoding the first 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 glycosylation consensus sequences (Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919). Transcription/translation of these fusion vectors in rabbit reticulocyte lysate +/- dog pancreatic microsomes followed by SDS-polyacrylamide gel electrophoresis defined the presence of signal anchor sequences in HK-M0 by glycosylation and stop transfer sequences in HK-M1 by inhibition of glycosylation. Six out of the seven putative transmembrane domains had membrane insertion signals, but no membrane insertion activity was found for the H3 segment in these vectors. To test the effect of specific upstream and downstream sequences 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 glycosylation 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 segment acted as a stop transfer sequence only when the whole N terminus and H3 were followed by the positive charges in the cytoplasmic loop between H3 and H4. The activity of H6 as a signal anchor sequence depended on preceding positive charges. These translation data using two types of fusion vectors establish a seven-transmembrane folding model using only in vitro translation for the CCK-A receptor beginning with two signal anchor sequences and then alternating stop transfer and signal anchor insertions. Positive charges between H1 and H2, H3 and H4, and H5 and H6 function as cytoplasmic anchors in the membrane folding of this receptor.