Molecular Endocrinology, Vol 10, 420-431, Copyright © 1996 by Endocrine Society

ARTICLES

Molecular cloning of human p48, a transient component of progesterone receptor complexes and an Hsp70-binding protein

V Prapapanich, S Chen, SC Nair, RA Rimerman and DF Smith
Department of Pharmacology, University of Nebraska Medical Center, Omaha 68198, USA.

A 48-kDa protein (p48) that transiently associates with progesterone receptor during cell-free assembly in rabbit reticulocyte lysate was isolated by two-dimensional gel separation. Tryptic peptide sequences were generated and used to develop an antipeptide antiserum recognizing p48 by Western immunostaining, and this antiserum was used to monitor purification of native p48 from reticulocyte lysate. Eight mouse monoclonal antibodies capable of immunoprecipitating vertebrate p48 were generated. These monoclonal antibodies served as probes to clone ten p48 cDNAs from a HeLa cDNA expression library. One of the cloned cDNAs was sequenced in its entirety and codes for a 369-amino acid protein (calculated Mr = 41,324). Northern blot analysis of RNA from multiple human tissues suggest that p48 may be ubiquitously expressed. Expression of human p48 cDNA in vitro yielded a product that comigrated with rabbit p48 by SDS-PAGE and associated with progesterone receptor in a similar manner. Immunoprecipitation of p48 complexes revealed a common association of p48 with hsp70 and, to a lesser extent, with hsp90 and p60. Thus, it appears that p48 is a novel component of the cytoplasmic molecular chaperone machinery.

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