Functional Analysis of Recombinant Mutants of Maxadilan with a PAC1 Receptor-expressing Melanophore Cell Line

• Published in: The Journal of biological chemistry 2006-06, Vol.281 (24), p.16197-16201
• Main Authors: Reddy, Vemuri B., Iuga, Aurel O., Kounga, Kounga, Lerner, Ethan A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cell Line; DNA Mutational Analysis; Insect Proteins - genetics; Insect Proteins - metabolism; Lutzomyia longipalpis; Melanophores - metabolism; Molecular Sequence Data; Pituitary Adenylate Cyclase-Activating Polypeptide - metabolism; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I - metabolism; Recombinant Proteins - chemistry; Sequence Homology, Amino Acid; Xenopus laevis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M509429200

Maxadilan, a 61-amino-acid vasodilatory peptide, was initially isolated from the salivary glands of the sand fly Lutzomyia longipalpis. Although its primary sequence has no homology to that of pituitary adenylate cyclase-activating peptide, maxadilan is an agonist for the PAC1 receptor. A total of 58 substitution and deletion mutants was engineered in an effort to determine which residues were important for receptor activation. The mutants were characterized functionally using an assay based on pigment granule translocation in PAC1-expressing Xenopus laevis melanophores. Substitution of charged residues and proline 43 could alter (but not eliminate) the agonist activity of the mutants. In contrast, we found that several multiple substitution mutants of the predicted β-strand threonine residues became antagonists at the PAC1 receptor. The results suggest that these threonine residues are cooperatively involved in PAC1 activation.