The disulfide-bonded loop of chromogranins, which is essential for sorting to secretory granules, mediates...

Chromogranins A and B, two widespread neuroendocrine secretory proteins, contain a homologous N-terminal disulfide-bonded loop that is required for sorting to secretory granules. Here we have investigated the role of this loop in the oligomerization of chromogranin A. Reduction of the disulfide bond or the addition of an excess of an N-terminal chromogranin A fragment containing the loop (CgA1-60) resulted in the dissociation into monomers of the chromogranin A dimer found at pH 7.4 and 6.4 and of the chromogranin tetramer found at pH 5.4. The addition of an excess of a synthetic peptide corresponding to the conserved C-terminal domain of chromogranin A (CgA406-431) had no effect on the chromogranin dimers at pH 7.4 and 6.4 and resulted in the dissociation of the chromogranin A tetramers at pH 5.4 into dimers. Fluorescence energy transfer experiments using fluorescently labeled CgA1-60 showed that the N-terminal disulfide-bonded loop has a high affinity for homodimerization (KD = 20 nM at pH 6.4), which was sufficient to mediate dimerization of full-length chromogranin A. Association and dissociation of loop-mediated chromogranin A dimerization approached completion within a few seconds. Our results imply that chromogranin A homodimerizes shortly after synthesis in the endoplasmic reticulum and that the loop-mediated homodimeric state is an essential prerequisite for its sorting, in the trans-Golgi-network, to secretory granules.