Characterization of two patched receptors for the vertebrate hedgehog protein family

Abstract

The multitransmembrane protein Patched (PTCH) is the receptor for Sonic Hedgehog (Shh), a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis. Current models suggest that binding of Shh to PTCH prevents the normal inhibition of the seven-transmembrane-protein Smoothened (SMO) by PTCH. According to this model, the inhibition of SMO signaling is relieved after mutational inactivation of PTCH in the basal cell nevus syndrome. Recently, PTCH2, a molecule with sequence homology to PTCH, has been identified. To characterize both PTCH molecules with respect to the various Hedgehog proteins, we have isolated the human PTCH2 gene. Biochemical analysis of PTCH and PTCH2 shows that they both bind to all hedgehog family members with similar affinity and that they can form a complex with SMO. However, the expression patterns of PTCH and PTCH2 do not fully overlap. While PTCH is expressed throughout the mouse embryo, PTCH2 is found at high levels in the skin and in spermatocytes. Because Desert Hedgehog (Dhh) is expressed specifically in the testis and is required for germ cell development, it is likely that PTCH2 mediates its activity in vivo. Chromosomal localization of PTCH2 places it on chromosome 1p33-34, a region deleted in some germ cell tumors, raising the possibility that PTCH2 may be a tumor suppressor in Dhh target cells.

Figure 1

Amino acid sequence homology between human PTCH and PTCH2. The predicted amino acid sequence of human PTCH2 is aligned with PTCH (31). Gaps introduced for optimal alignment are shown by dashes. Identical amino acids are boxed. The transmembrane domains are highlighted. The conserved glycosylation site is indicated by asterisks.

Figure 2

PTCH2 interacts with hedgehogs and SMO. (a) Immunostaining of COS-7 cells transfected with PTCH or PTCH2 and labeled with Shh-IgG or Dhh-IgG (red), and antibody directed against the Flag epitope (green). No labeling was detected in cells transfected with a control vector (data not shown). (b) Competition binding of recombinant murine ¹²⁵I-Shh to 293 cells overexpressing hPTCH or hPTCH2. There was no detectable binding to mock-transfected cells (data not shown). (c) Coimmunoprecipitation of epitope-tagged PTCH or PTCH2 with epitope-tagged SMO. Immunoprecipitation was performed with antibodies to the Flag-tagged PTCH and analyzed by Western blotting on a 6% acrylamide gel with antibodies to the Myc-tagged SMO. Protein complexes can be detected with both PTCH and PTCH2. PTCH and PTCH2 express at similar levels as shown by immunoprecipitation by using antibodies to the Flag tag and by Western blot by using the same anti-Flag antibody.

Figure 3

Northern blot analysis of mouse PTCH and PTCH2. Northern blots containing RNA from multiple mouse tissues, as indicated, were hybridized with a cDNA fragment corresponding to the 3′ untranslated region of mPTCH or mPTCH2.

Figure 4

In situ hybridization analysis of PTCH, PTCH2, and Fused. Emulsion autoradiographs after in situ hybridization of sagittal sections through embryonic day 18 (E18) mouse for PTCH (a) or PTCH2 (b) probes, transverse sections through E18 wild-type or SMO-M2 transgenic mouse skin for PTCH (c and e) and PTCH2 (d and f), and sections of adult mouse testis for PTCH (g), PTCH2 (h and k), Fused (i and l), and Dhh (j). [Bar = 2.1 mm (a and b); 0.065 mm (c– f, j); 0.12 mm (h and i); 1.0 mm (k and l).] bn, bone; gt, gut; sk, skin; th, teeth; vib, vibrissae; ep, epidermis; de, dermis; fol, hair follicle.