Bovine thrombospondin-2: Complete complementary deoxyribonucleic acid sequence and immunolocalization in the external zones of the adrenal cortex

Given the variety of biological functions in the adrenal cortex that are co ntrolled by ACTH, we hypothesized that some extracellular proteins act as b iological relays for this systemic hormone. One candidate protein [corticot ropin-induced secreted protein (CLSP)] was purified from the conditioned me dium of bovine adrenocortical cells on the basis of a 5- to 14-fold increas e in its synthesis after the addition of ACTH. We report here the cloning o f overlapping complementary DNAs that span the sequence encoding the full-l ength protein (1170 amino acids). The de duced CISP protein sequence is 89% identical to that of human thrombospondin-2 (TSP2), but only 61% identical to that of bovine TSP1, confirming that CISP is the bovine ortholog of TSP 2. The bovine TSP2 sequence aligned perfectly with human, mouse, and chicke n TSP2 sequences, except for a gap of 2 amino acids located in a linker reg ion. AU 58 cysteine residues that are conserved in other species were prese nt in the bovine sequence as well as most of the functional domains. Most e ndocrine tissues (adrenal cortex, testis, ovary, and placenta) appeared to express TSP2, as determined by Western blot analysis. The highest levels of TSP2 protein were found in the adrenal cortex, followed by the heart, sple en, brain, and kidney. A differential extent of N-glycosylation or tissular proteolytic maturation may be responsible for the mol art differences obse rved between bovine TSP2 detected in the medium from primary cultures and t hat in fresh tissue extracts. The immunohistochemical analysis of the distr ibution of TSP2 in the bovine adrenal gland revealed that the protein is mu ch more abundant in the external zones (zona glomerulosa and zona fascicula ta) than in the internal reticularis zone, a pattern similar to that report ed for ACTH receptors. This distribution clearly suggests that TSP2 is a ca ndidate relay protein for a subset of ACTH actions in the adrenal cortex.