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Structure and expression of the cAMP cell-surface receptor
1Laboratory of Cellular and Developmental Biology, NIDDK, Bethesda, MD 20892.
Abstract:
Using antibodies specific for the 3',5'-cyclic AMP (cAMP) cell surface receptor of Dictyostelium discoideum, we have screened lambda gtll expression libraries and isolated a series of cDNAs derived from cAMP receptor mRNA during early development. The identity of the cDNA clones was verified by multiple criteria: 1) beta-galactosidase fusion proteins synthesized by isolated cDNA clones stain intensely with cAMP receptor directed antiserum, 2) these fusion proteins affinity purify antibodies specific for the cAMP receptor, 3) the cDNA probes hybridize to a 2 kb mRNA whose change in relative level of abundance during development parallels that of receptor mRNA as assayed by in vitro translation, 4) the 2 kb mRNA size equals that of receptor mRNA as determined by in vitro translation of size fractionated poly (A)+ RNA, and 5) RNA transcribed in vitro from cDNAs containing the entire protein-coding region produces a polypeptide by in vitro translation with an apparent molecular weight in close agreement with that of nascent cAMP receptor protein produced by in vitro translation of cellular RNA. The DNA sequence predicts an open reading frame of 392 amino acids. The deduced amino acid sequence contains seven domains enriched in hydrophobic residues. A model is proposed in which the cAMP cell-surface receptor traverses the lipid bilayer seven times in a pattern similar to that of other receptors, such as rhodopsin, which interact with G-proteins. The structural similarities suggest a gene family of related surface receptors from such evolutionarily diverse species as Dictyostelium, yeast, and mammals.
Insights
Researchers identified the cyclic AMP (cAMP) cell surface receptor in Dictyostelium discoideum using specific antibodies. This discovery provides insights into early development and potential gene family relationships across species.
Area of Science:
- Molecular Biology
- Developmental Biology
- Cell Biology
Background:
- The cell surface receptor for cyclic AMP (cAMP) plays a crucial role in the early development of Dictyostelium discoideum.
- Understanding the molecular characteristics of this receptor is essential for deciphering developmental signaling pathways.
Purpose of the Study:
- To isolate and characterize the complementary DNAs (cDNAs) encoding the Dictyostelium discoideum cAMP cell surface receptor.
- To elucidate the structural features and potential membrane topology of the cAMP receptor.
Main Methods:
- Screening of lambda gt11 expression libraries using antibodies against the cAMP receptor.
- Verification of cDNA clone identity through beta-galactosidase fusion protein analysis, antibody purification, and nucleic acid hybridization.
- In vitro transcription and translation of isolated cDNAs.
- DNA sequencing and analysis of the deduced amino acid sequence.
Main Results:
- Isolation of multiple cDNAs corresponding to the cAMP receptor mRNA.
- Confirmation that the 2 kb mRNA hybridizes with cDNA probes and its abundance changes during development.
- The deduced amino acid sequence predicts a 392-amino acid protein with seven hydrophobic domains.
- A model suggesting seven transmembrane passes, similar to G-protein coupled receptors like rhodopsin.
Conclusions:
- The study successfully identified and characterized the Dictyostelium discoideum cAMP cell surface receptor at the molecular level.
- Structural analysis suggests the receptor is a seven-transmembrane protein, indicative of a conserved structural motif in cell surface signaling.
- The findings suggest a potential gene family of related surface receptors conserved across diverse species, including yeast and mammals.