The recently purified and sequenced parathyroid hormone-related proteins (PTHrPs), responsible for humoral hypercalcemia of malignancy, have been shown to interact with parathyroid hormone (PTH) receptors in bone and kidney. PTHrPs are produced by a wide variety of normal tissues as well as tumors. Abundant evidence is accumulating to support the hypothesis that the normal function of PTHrP lies in the autocrine/paracrine realm serving as a regulator of cellular differentiation and proliferation. We have previously identified the presence of PTHrP binding and signalling mechanisms in benign keratinocytes (HK) and their malignant counterpart, squamous carcinoma cells (SqCC). We have not accumulated preliminary evidence for the existence of two classes of putative cell surface receptors for PTHrPs on HK and SqCC. One has specificity for aminoterminal PTHrPs, and the other for a midregion PTHrP. The major objectives of this proposal therefore are to clone cDNAs encoding these PTHrP receptors and to characterize the expressed receptors. This will allow for further study of their mechanism of action, and will provide us with the tools for more definitive studies into the biological action of PTHrPs in squamous epithelial cells, which is the ultimate goal of this project. The following Specific Aims are identified: 1) To molecularly clone and pharmacologically characterize unique N-terminal PTHrP receptors on keratinocytes and squamous carcinomas. The presence of sensitive intracellular calcium transients and the lack of adenylate cyclase responses to (Tyr36)hPTHrP(1-36)NH2 and hPTH(1-34) in squamous cells suggest that this receptor differs from the "classical" PTH receptors expressed in bone and kidney. Northern blots using a PTH receptor cDNA probe indicate that squamous cells do not express the 2.5 kb transcript which is seen in bone and kidney; instead, a predominant 1 kb transcript is visualized along with several larger transcripts. We propose to further characterize these transcripts with respect to region of homology to the PTH receptor using Norther and RNase protection analysis, and PCR mapping. cDNA cloning will be pursued via PTH receptor cDNA hybridization and PCR strategies. 2) To clone and pharmacologically characterize a unique mid-region PTHrP receptor mediating the hPTHrP(67- 86)NH2 cytosolic calcium signaling in human squamous carcinoma cells. A unique midregion PTHrP is secreted by HK and SqCC, and preliminary studies indicate that two SqCC lines demonstrate sensitive intracellular calcium response to hPTHrP(67-86)NH2. We propose to further characterize these signalling mechanisms. The presence of calcium as an expression cloning system to clone full-length cDNAs encoding this receptor.