Heterogeneity of messenger RNA that encodes the rat insulin receptor is limited to the domain of exon 11. Analysis by RNA heteroduplex mapping, amplification of cDNA, and in vitro translation.
Structural isoforms of the insulin receptor that occur in various tissues have been postulated to be involved in certain actions of insulin in target cells. To determine whether these insulin-receptor subtypes are caused by alterations in the receptor primary structure, we used RNA heteroduplex mapping and amplification of cDNA to detect variation in the coding region of insulin-receptor mRNA from 5 rat tissues. A complete series of overlapping antisense [32P]RNA probes was prepared from plasmids containing segments of a full-length rat insulin-receptor cDNA, and probes were hybridized individually in solution with polyadenylated RNA from rat brain, kidney, liver, skeletal muscle, and spleen. After ribonuclease digestion, probe fragments were analyzed by denaturing gel electrophoresis. Tissue-specific cleavage of the mRNA:RNA probe heteroduplex, attributable to sequence mismatch, was detected only for a single probe covering the distal alpha-subunit, as expected for the known alternative splicing of rat insulin-receptor mRNA in this region. No evidence for additional heterogeneity of the receptor mRNA coding region was observed in the 5 tissues studied either by RNA heteroduplex mapping or, in some areas, by regional amplification of insulin-receptor cDNA. Cell-free translation of size-fractionated polyadenylated RNA was used to further demonstrate that each of the major insulin-receptor mRNA size classes in rat liver contained both forms of the alternatively spliced mRNA transcripts and produced two insulin-proreceptor polypeptides. These results suggest that heterogeneity of the insulin-receptor mRNA coding region affecting the receptor primary structure is limited to the distal alpha-subunit near the subunit cleavage site.(ABSTRACT TRUNCATED AT 250 WORDS)
Goldstein
B J
BJ
Research Division, Joslin Diabetes Center, Boston, Massachusetts.
Dudley
A L
AL
D000818:Animals; D016375:Antisense Elements (Genetics); D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005091:Exons; D014644:Genetic Variation; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics* / Q000302:isolation & purification; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000235:genetics*; D015183:Restriction Mapping
D016375:Antisense Elements (Genetics); D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D015347:RNA Probes; D012333:RNA, Messenger; D004247:DNA; D011972:Receptor, Insulin