Molecular cloning, localization and characterization of a 40-kDa catecholamine-regulated protein

We have previously described catecholamine-regulated proteins of molecular masses 47, 40 and 26 kDa (CRP47/40/26). In mammals, these proteins are dete cted only in brain and have been implicated as playing a role in dopaminerg ic neurotransmission. In this report, we have cloned the cDNA encoding CRP4 0 from bovine brain. Analysis of the predicted amino acid sequence revealed that the CRP40 product contains an hsp70 motif and shares homology with he at shock protein hsp70. Immunolocalization studies using mAbs to dopamine s how that it colocalizes with CRP40 in the vesicles of dopaminergic neurobla stoma SH-SY5Y cells. The constitutive expression of CRP40 was increased by exposure to heat shock similar to inducible heat-shock protein hsp70 in SH- SY5Y cells. Dopamine significantly modulated the levels of CRP40, whereas, the expression of hsp70 remained unchanged upon dopamine treatment of these cells. Moreover, CRP40 is able to prevent the thermal aggregation of lucif erase in vitro, similar to hsp70, suggesting that CRP40 encodes a dopamine- inducible protein with properties similar to heat-shock proteins. The immun ofluorescence analyses show that in SH-SY5Y cells, CRP40 translocates to th e nucleus during dopamine-induced apoptosis. These results suggest that CRP 40 could play a protective role against the harmful effects of catecholamin e metabolites.