The mouse gastric H,K-ATPase beta subunit. Gene structure and co-ordinate expression with the alpha subunit during ontogeny.

G P Morley,J M Callaghan,J B Rose,B H Toh,P A Gleeson,I R Van Driel

doi:10.1016/s0021-9258(18)48410-4

Abstract

The gastric H,K-ATPase (EC 3.6.1.3) is responsible for acid secretion into the stomach and is composed of two subunits, alpha and beta. The structure of the gene encoding the mouse beta subunit and the expression of both subunits during ontogeny are reported. The gene spans approximately 12 kilobase pairs and contains 7 exons. The positions at which introns interrupt the coding regions of the mouse H,K-ATPase beta subunit and mouse Na,K-ATPase (EC 3.6.1.37) beta 2 subunit genes are identical. The alternative beta subunit isoform of the Na,K-ATPase, beta 1, has a similar but not identical gene structure. Primer extension and S1 nuclease analysis of RNA isolated from mouse stomachs aged between 2 and 25 days indicated that major transcription-initiation sites are between 22 and 25 base pairs 5' of the translation initiation site at all ages. The expression of the H,K-ATPase alpha and beta subunit genes during ontogeny (day 1-40) was found to be co-ordinated. Protein levels of both the ATPase alpha and beta subunits were very low until day 15 and then increased to adult levels by day 30. In any mucosal cell throughout ontogeny, expression of the beta subunit gene invariably coincided with the expression of the alpha subunit gene. Cells detected by anti-H,K-ATPase beta subunit antibodies in sections from 10- and 30-day-old mice all had typical morphology of parietal cells and were arranged in glandular structures. Co-ordinate expression of the two subunit genes suggest that the regulatory mechanisms will be similar and that the beta subunit may be required for localization and function of the catalytic alpha subunit.

Highlights

@. for acid secretion into the stomach anids composed of parietal cells, thesemembrane structures appear as cytotwo subunits,a and The structureof the geneencod- plasmic tubulovesicles
The alternative @ subunit isoform of the Na,KATPase, @1h,as a similar butnot identical gene structure.Primerextensionand SI nuclease analysis of RNA isolated from mouse stomachs aged between 2 secrete acid upon stimulation is probably due to changes in K+ and C1- ion conductance and not due to increased levels of gastric H,K-ATPase protein
The enzyme is composed of two subunits: a 95-100-kDa a subunit (Shull and Lingrel, 1986;Forte and Soll, 1989;Rabon and Reuben, 1990), and a p subunit that appears as a heterand 25 days indicatedthat major transcription-initia- ogeneous species of 60-90-kDa when analyzed by sodium tion sites are between 22 and 25 base pairs 5’ of the dodecyl sulfate-polyacrylamide gel electrophoresis

Summary

EXPERIMENTAL PROCEDURES

Antibodies ana' Animals-Monoclonal antibodies directed against the H,K-ATPase subunits 2B6 ( B subunit) and 1H9 (a subunit) have been described, and both are IgGl (Mori et al.,1989). Transcription Initiation Site Mapping-Primer extension analysis of RNA was performed with two primers of 49 and 69 nucleotides in length that were prepared and 32P-labeledas described (Sambrook et al, 1989).Briefly, two oligonucleotides (+39 to +63 and +64 to +83) were separately annealed to a single-stranded template generated from a phagemid vector, pBluescript KS-, containing the 3.2-kb BamHI-XbaI fragment ofXmB3 (a XbaI site is located at -1059 and a BamHI site is located 2.1 kbp 3' of the translation initiation site). Sections were incubated with undiluted 1H9 hybridoma supernatant, washed as before, and incubated with tetramethylrhodamine isothiocyanate (TR1TC)-labeled rabbit anti-mouse immunoglobulins (Dakopatts, Denmark; catalog no.R270, diluted 1:lOO in PBS). Sections were incubated with pooled sera from patients with pernicious anemia (diluted 1:lO in PBS) followed by FITC-labeled sheep anti-human immunoglobulins (Welcome Diagnostics, Dartford, United Kingdom; diluted 1:40 in PBS). After a final wash inPBS, the sections were mounted and examined by epifluorescence microscopy as above

Control incubations were performed with monoclonal antibody

RESULTS

Ph G h r

HUM PI

This data strongly suggest the following genealogy for the s

Plenum Press