Localization and topology of ratp28, a member of a novel family of putative steroid-binding proteins

doi:10.1016/S0167-4838(00)00188-6

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 1543, Issue 1, 30 November 2000, Pages 123-130

Biochimica et Biophy...

https://doi.org/10.1016/S0167-4838(00)00188-6 Get rights and content

Abstract

We have cloned ratp28, a membrane protein from rat liver homologous to the previously described hpr6.6, a putative steroid-binding protein in humans. Ratp28 has a type II topology as determined by protease digestion experiments on intact and detergent-solubilized membranes. Subcellular fractionation by sucrose density centrifugation revealed a distribution for ratp28 identical to Bip as a marker for membranes of the endoplasmic reticulum. In these experiments no association was found with markers for Golgi or plasma membranes, indicating that ratp28 is localized to the endoplasmic reticulum.

Introduction

Steroids like progesterone act via different mechanisms inside their respective target cells. The classical mode of action includes penetration of the steroids through the plasma membrane and binding to an intracellular cytosolic receptor. Subsequently, these steroid-receptor complexes are translocated into the nucleus where they function as transcription factors [1]. Recently it has been shown that in addition to modulating transcription, which is a rather ‘slow’ way of action (>7 min [2]), steroids also have more rapid, non-genomic effects. For example, progesterone was shown to affect Ca²⁺ influx and Cl⁻ efflux during the acrosomal reaction of human sperms [3], [4]. Furthermore, progesterone seems to influence the Ca²⁺ levels in hepatocytes [5]. Grazzini et al. [6] demonstrated a direct, non-genomic effect of progesterone on the plasma membrane bound uterine oxytocin receptor. Moreover, approaches using radioactively labeled progesterone as a ligand led to the identification of several proteins with putative progesterone-binding properties from different sources and different species, including membranes from mouse brain [7] or porcine liver membranes [8]. This has led to the assumption that membrane associated steroid receptors probably exist. Falkenstein et al. have cloned cDNA from porcine vascular smooth muscle cells coding for a protein that is able to bind progesterone [9]. This protein has an apparent molecular mass of 28 kDa and behaves like an integral membrane protein [10]. It was suggested that it is localized to membranes of the endoplasmic reticulum or the Golgi complex [11]. More recently a human homologue of the 28 kDa protein, hpr6.6, and a closely related human protein, dg6, with significant homology were identified, indicating that these polypeptides belong to a family of proteins [12].

Here we describe the molecular characterization of ratp28. We show that this protein belongs to the family of proteins with putative progesterone-binding properties. We suggest that ratp28 has a type II topology. Furthermore we have localized this protein by subcellular fractionation to membranes of the endoplasmic reticulum rather than to Golgi membranes.

Section snippets

Antibodies

Anti-peptide antibody directed against the N-terminus of ratp28 was raised in rabbits by coupling the peptide EDVVATGADPSELE to keyhole limpet hemocyanin (KLH) according to standard protocols [13]. Antibodies were further purified by affinity chromatography using peptide-coupled Sepharose (Pharmacia, Germany).

Anti-DPP IV antibodies and anti-protein disulfide isomerase antibodies (MAC 256) were generously provided by Prof. Dr. Werner Reutter, Berlin and by Dr. Ingrid Haas, Heidelberg,

Results

Isolated membrane fractions enriched in Golgi membranes were isolated from rat liver by isopycnic centrifugation. Membrane proteins, obtained by alkaline treatment of these membranes, were further analyzed by subsequent SDS-gel electrophoresis. The NH₂-terminal amino acid sequences of two proteins with apparent molecular masses of 23 and 28 kDa, respectively, were determined by microsequencing of the corresponding bands (data not shown). The 23 kDa protein was identified as p23, a transmembrane

Discussion

We have cloned ratp28, the rat homologue of the human transmembrane protein hpr6.6 and of several other proteins of different species. Computer analysis, using the algorithm of Kyte and Doolittle, reveals that ratp28 contains a hydrophobic domain near its N-terminus typical for transmembrane proteins. This is confirmed by the fact that ratp28 is present in pellet fractions after carbonate treatment of microsomal membranes, and that the porcine homologue was extracted in the detergent phase of

Acknowledgements

We are indebted to Prof. W. Reutter, Berlin, for generously providing antibodies against DPP IV, and to Dr. I. Haas, Heidelberg, for providing antibodies against Bip and PDI. Furthermore, we thank S. Auerbach and S. Wegehingel for their helpful assistance, Kirk Fischer and Nicola Saunders for their constructive criticism.

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Localization and topology of ratp28, a member of a novel family of putative steroid-binding proteins

Abstract

Introduction

Section snippets

Antibodies

Results

Discussion

Acknowledgements

Cell. Signal.

Mol. Cell. Endocrinol.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

Steroids

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Trends Biochem. Sci.

J. Mol. Biol.

Nature

Nucleic Acids Res.

Nature