IDENTIFICATION OF ALTERNATIVELY SPLICED MESSENGER-RIBONUCLEIC-ACID ENCODING TRUNCATED GROWTH HORMONE-RELEASING HORMONE-RECEPTOR IN HUMAN PITUITARY-ADENOMAS

The expression of GHRH receptor (GHRH-R) messenger ribonucleic acid (m RNA) was studied in 22 pituitary adenomas and 2 normal anterior pituit aries. Northern blot analysis revealed that GHRH-R mRNA were expressed in all 14 GH-producing adenomas, 1 of 3 ACTH-producing adenomas, the 1 PRL-producing adenoma, 2 of 4 nonfunctioning adenomas, and the 2 nor mal anterior pituitaries. Their expression levels varied among GH-prod ucing adenomas and were relatively low in;GH-nonproducing adenomas. In addition to the major transcript with a molecular mass of 2.0 kilobas es (kb), the transcripts were identified at 2.8 and 4.5 kb in some GH- producing adenomas. To examine the structural variations in GHRH-R mRN A in pituitary adenomas, we amplified the complementary DNA fragment e ncompassing the region from the third cytoplasmic loop to the sixth tr ansmembrane domain of GHRH-R. This region was selected because this re gion of the G protein-coupled receptor has been known to interact with G protein. Two amplified fragments with the molecular masses of 250 a nd 810 base pairs were identified by the reverse transcriptase-polymer ase chain reaction method. The nucleotide sequence of a smaller fragme nt, which was the expected size, revealed that no mutations were found in this region in 10 GH-producing adenomas examined. However, a large r fragment contained the currently unidentified insertion. Compared wi th the genomic DNA sequence, this insertion was found to be generated through alternative splicing. In addition, this variant form contained the premature stop codon in-frame, indicating that it encodes the tru ncated GHRH-R. This insertion-specific probe could hybridize with 2.8- and 4.5-kb species of GHRH-R mRNA on Northern blot analysis, and thes e transcripts were expressed mainly in GH-producing adenomas. Finally, study of cell transfection and cAMP measurement revealed that this tr uncated GHRH-R was unable to transmit GHRH signals. These results sugg est that some GK-producing adenomas preferentially express the truncat ed GHRH-R as a nonfunctioning receptor through alternative splicing.