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Molecular cloning, chromosomal localization of human peripheral-type benzodiazepine receptor and PKA regulatory subunit type 1A (PRKAR1A)-associated protein PAP7, and studies in PRKAR1A mutant cells and tissues¹

JUN LIU^*, LUDMILA MATYAKHINA, ZEQIU HAN^*, FABIANO SANDRINI, THALIA BEI, CONSTANTINE A. STRATAKIS and VASSILIOS PAPADOPOULOS^*,2

^* Division of Hormone Research, Departments of Cell Biology, Pharmacology and Neuroscience, Georgetown University Medical Center, Washington, D.C., USA; and
Unit on Genetics and Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA

²Correspondence: Division of Hormone Research, Departments of Cell Biology, Pharmacology and Neuroscience, Georgetown University Medical Center, Washington, DC 20057, USA. E-mail: papadopv{at}georgetown.edu).

SPECIFIC AIMS

A mouse protein that interacts with the peripheral-type benzodiazepine receptor (PBR) and cAMP-dependent protein kinase A (PKA) regulatory subunit RI (PRKAR1A), named PBR- and PKA-associated protein 7 (PAP7), was identified and shown to be involved in hormone-induced steroid biosynthesis. The present studies were undertaken to 1) characterize the human homologue of the mouse PAP7 cDNA, isolate the human PAP7 gene, and determine its chromosomal localization; 2) investigate the expression of the PAP7 mRNA and protein in human tissues and cells; 3) based on the PAP7 expression pattern, identify a human pathology where this protein might play a role and help elucidate its physiopathological function.

PRINCIPAL FINDINGS

1. The rate-determining step in the hormone-stimulated steroid biosynthesis is transport of the precursor cholesterol from intracellular sources into the mitochondria
In search of the structural elements participating in this mitochondrial uptake and transfer of cholesterol, we identified the peripheral-type benzodiazepine receptor (PBR), a high-affinity drug ligand and cholesterol binding protein. In steroidogenic cells, mitochondrial PBR ligand binding capacity and topography are regulated by cAMP, the cAMP-dependent PKA, and cytosolic factor(s). Using the yeast two-hybrid technique and a mouse testis cDNA library and PBR as bait, we recently identified a protein interacting with PBR with a tissue distribution close to that of PBR. When screening a human lymphocyte library with the regulatory subunit RI of PKA (PRKAR1A) as bait, a partial clone encoding for a protein homologous to the previously isolated mouse protein interacting with PBR with in vivo selectivity for PRKAR1A was isolated. This protein was named PAP7 for PBR and PKA-associated protein 7. This finding suggested that PAP7 might possibly mediate the targeting of PRKAR1A to mitochondria rich in PBR, where a cAMP-dependent event would occur leading to cholesterol uptake and transport into the mitochondria.

A full-length PAP7 cDNA was isolated (GenBank accession #AY150218) and revealed an ORF encoding a 528 amino acid protein with a calculated molecular mass of 60.5 kDa. By sequence motif analysis, human PAP7 was found to have N-glycosylation, PKC phosphorylation, casein kinase II phosphorylation, tyrosine kinase phosphorylation, and N-myristoylation sites as well as arginine, glutamine, glutamic acid, and proline-rich regions. An acyl-CoA binding protein signature motif was identified. Searching the corresponding contigs from draft human genome database, PAP7 cDNA was found to be highly homologous with contig NT_004525. The human PAP7 gene was 48 kb in length and contained eight exons and seven introns. An initial homology search showed that this sequence had not been identified before. In a recent search, however, PAP7 was found to show high identity to the recently cloned human Golgi complex-associated protein 1 GOCAP1 amino acid sequence. Confocal microscopy in MA-10 Leydig cells indicated that PAP7 is localized both in Golgi and mitochondria. Human PAP7, mouse PAP7, and the endogenous PBR ligand, the polypeptide diazepam binding inhibitor (also known as acyl-CoA binding protein) show a 30% identity. The human PAP7 gene was mapped to chromosome 1q32-41 by fluorescence in situ hybridization.

2. Human PAP7 mRNA was found in most tissues and cell lines examined; steroidogenic tissues, such as testis, contained higher levels of PAP7 mRNA
PAP7 protein levels were also found to be high in adrenal, ovary, and testis by immunoblot analysis of tissue extracts (Fig. 1 ). PAP7 protein expression and distribution in different tissues were investigated in detail by immunohistochemistry. In agreement with the mRNA data, PAP7 was found in most tissues examined. PAP7 immunoreactivity was very strong in adrenal cortex, atrium, colon epithelial cells, esophagus, jejunum, renal cortex, testicular Leydig cells, vena cava, and ventricular muscle. Liver expressed a low level of PAP7 protein. In contrast to the high expression of mouse PAP, mRNA and protein in brain central and peripheral nervous system tissues (such as autonomic nerve, caudate nucleus, cerebella cortex, dura mater, peripheral nerve, spinal cord, and thalamus) did not show immunoreactivity for PAP7.

View larger version (32K): [in this window] [in a new window]   Figure 1. Immunoblot analysis of human PAP7 expression in different tissues. 50 µg total protein of each selected tissue was separated by SDS-PAGE and the 60 kDa PAP7 protein was detected using affinity-purified anti-PAP7 peptide antiserum. PAP7 is highly expressed in adrenal, ovary, skin, and testis.

Three observations encouraged us to look closer in the relationship of PAP7 to human adrenal pathology: 1) the high level of PAP7 protein expression in human adrenal cortex; 2) the recent finding that the gene encoding PRKAR1A, mapped to chromosome 17q22-24, is mutated in Carney complex (CNC), a multiple neoplasia syndrome caused by germline-inactivating PRKAR1A mutations (PRKAR1A-mut); 3) localization of PAP7 in several tissues targets of CNC such as pituitary, thyroid, skin, heart, and steroidogenic tissues, including the gonads and adrenal cortex. The latter is affected in CNC by primary pigmented nodular adrenocortical disease (PPNAD), a unique adrenal disorder characterized by ACTH-independent hypercortisolism that is stimulated by exogenous steroids. So far there has been no explanation for this unusual steroidogenic behavior. Immunohistochemistry of PPNAD specimens from patients carrying PRKAR1A-inactivating mutations revealed intense staining for PRKAR1A and PAP7 in PPNAD tissue surrounding the nodules, whereas nodular cells were either weakly stained in a nonhomogeneous manner or not stained at all (Fig. 2 ). For PRKAR1A, this is consistent with the lack of protein predicted by the allele carrying the PRKAR1A-inactivating mutation and the absence of normal allele in tumoral tissue, as predicted by loss-of-heterozygosity. Adrenal tissue surrounding the PPNAD nodules showed intense immunoreactivity for both PAP7 and PRKAR1A. For PAP7, this was an unexpected finding indicating the coordinated expression of these two proteins. The coordinated regulation of PRKAR1A and PAP7 expression seen in PPNAD tissues was further confirmed in two lymphoblastoid cell lines derived from patients with inactivating PRKAR1A mutations. These findings correspond well with data suggesting that CNC tumors carrying PRKAR1A-mut respond to cAMP stimulation with an increase in PKA activity compared with adrenal steroidogenic tissues that do not have PRKAR1A mutations. The specificity of this type of regulation in adrenal tumors with PRKAR1A-mut is supported by observations that there was no difference in PAP7 protein expression in cases of adrenal hyperplasia and no significant difference between normal and brain, breast, and colon tumor PAP7 mRNA levels. The specificity of PRKAR1A localization is based on the observation that PKA-RII subunit expression is not significantly altered in PPNAD samples and does not correlate with PAP7 localization and expression.

View larger version (97K): [in this window] [in a new window]   Figure 2. Immunohistochemical staining of PPNAD nodules and the surrounding cortex. a, d, g, i, m) Control (hyperplasia); b, e, h, k, n) PPNAD mother; c, f, i, l, o) PPNAD-daughter. a–c) Anti-PAP7; d–f) anti-PBR; g–i) anti-StAR; j–l) anti-PRKAR1A; m–o) anti-PRKARII. p, q) Magnified picture of PAP7 staining in PPNAD mother.

CONCLUSIONS AND SIGNIFICANCE

We previously demonstrated that 1) overexpression of the full-length PAP7 increased hormone-induced steroid production, 2) overexpression of a partial PAP7, which includes the PBR and PRKAR1A binding domains, inhibited hormone-stimulated cholesterol transport and steroid synthesis, and 3) treatment of steroidogenic cells with oligonucleotides antisense to PAP7 inhibited the hormone-stimulated steroid formation. These results suggested that PAP7 is a functional element of the hormone-induced signal transduction cascade, where it would function as an adaptor protein allowing for the compartmentalization of cAMP and/or targeting of the PRKAR1A to its site of action at the mitochondria, leading to increased cholesterol transport and steroidogenesis.

We report here cloning and characterization of the human gene for PAP7 and show that, like in the mouse, human PAP7 is highly expressed in steroidogenic tissues. We report that PAP7 is tightly linked in its expression profile with PRKAR1A in PPNAD tissues. PAP7 message and protein levels were decreased in PPNAD nodules and PRKAR1A-mut; high levels of PRKAR1A and PAP7 proteins were seen in the adrenal tissue surrounding the nodules. We believe that these findings may offer an insight into understanding both the cause of tumor formation in PPNAD and the paradoxical responses of its hypercortisolism. Considering that PRKAR1A may function as a tumor suppressor, the presence of PRKAR1A-mut in PPNAD would lead to tumor formation, which over time will expand within the tissue. In the adrenal tissue surrounding the nodule the presence of even relatively low cAMP levels would result in an amplified activation of the PKA, as previously shown. Targeting of the residual PRKAR1A by increased amounts of PAP7 in the right subcellular compartment (i.e., mitochondria) would induce and sustain steroid formation, albeit in a dysregulated manner with regard to cAMP and other stimuli, leading to "paradoxical" responses. Figure 3 shows a schematic representation of the regulation of cortisol synthesis and tumor growth in PPNAD.

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic representation of the regulation of cortisol synthesis and tumor growth in PPNAD mediated by the PRKAR1A and PAP7 expression levels.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1066fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-1066fje

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