Our research program is concerned with the analysis, synthesis, expression, and structure-function relationships of peptide and protein hormones, with current emphasis on the structure and function of the angiotensin II (Ang II) and gonadotropin-releasing hormone (GnRH) receptors.

Synthesis of Fluorescent Gonadotropin-Releasing Hormone Agonist
Morell, Chen H-C
Recently, we completed the milligram-scale synthesis and purification of a sulforhodamine 101 (Texas Red) conjugate of [D-Lys6, des Gly10amide]GnRHethylamide at e-amino group of D-Lys6. The reaction of [D-Lys6, des- Gly10amide]GnRHethylamide with Nsucci-nimidyl ester of sulforhodamine 101 yielded five components as revealed by reverse-phase high-performance liquid chromatography in which the major component was characterized as the mono-sulforhodamine 101 acylated [D-Lys6, des Gly10amide]GnRHethylamide at eamino group of D-Lys6. As revealed in confocal microscopy, this major conjugate is biologically active as a consequence of its ability to bind to the GnRH receptor and to internalize in the same manner as the GnRH agonist.

Human Angiotensin II Type 1 Receptor Binding Proteins
Chen H-D, Chen H-C
In studies on the structure and function of the Ang II receptor, we demonstrated in vivo the association of hAT1 and the MHC class II–associated invariant chain Ii by yeast mating experiments and by co-localization at the plasma membrane of human embryonic kidney (HEK293) cells on confocal microscopy. For co-localization studies, we transfected HEK cells with both AT1-green fluorescent protein and Iired fluorescent protein fusion proteins. In this cell line, we demonstrated inhibition of hAT1 receptor internalization in response to Ang II stimulation in the presence of Ii on the plasma membrane. The manner in which the Ii chain interferes with agonist-induced endocytosis of the AT1 receptor is under further investigation.

Tandem Mass Spectrometric Sequencing of Phosphopeptides
Li, Wang, Chen H-C
We have also developed a chemical method that converts phospho-Ser and phospho-Thr to cysteic acid and b-methyl cysteic acid, respectively, for direct sequencing of the Ser and Thr phosphorylation sites by electron-spray ionization (ESI) tandem mass spectrometry. We modified and then, using an ESI-quadrupole ion trap mass spectrometer, sequenced five model phosphopeptides, including three synthetic PSer, P-Thr, or both P-Ser and P-Thrcontaining peptides; a protein kinase C–phos-phorylated bovine myelin basic protein residues 4-14 peptide; and a phosphopeptide derived from b-casein trypsin digests. Following incubation of P-Ser or P-Thr containing peptides with Na2SO3/NaOH, amino acid analysis revealed that 90 percent P-Ser and 80 percent PThr were converted to cysteic acid and bmethyl cysteic acid, respectively. The conversion can be carried out at 1 mM concentration of the peptide. Both cysteic acid and b-methyl cysteic acid residues in the sequence were shown to be stable and easily identifiable in a series of y and b ions under general conditions for tandem mass spectrometric sequencing applicable to common peptides.