Identification of a human type II receptor for bone morphogenetic protein-4 that forms differential heteromeric complexes with bone morphogenetic protein type I receptors.

Bone morphogenetic proteins (BMPs) comprise the largest subfamily of TGF-beta-related ligands and are known to bind to type I and type II receptor serine/threonine kinases. Although several mammalian BMP type I receptors have been identified, the mammalian BMP type II receptors have remained elusive. We have isolated a cDNA encoding a novel transmembrane serine/threonine kinase from human skin fibroblasts which we demonstrate here to be a type II receptor that binds BMP-4. This receptor (BRK-3) is distantly related to other known type II receptors and is distinguished from them by an extremely long carboxyl-terminal sequence following the intracellular kinase domain. The BRK-3 gene is widely expressed in a variety of adult tissues. When expressed alone in COS cells, BRK-3 specifically binds BMP-4, but cross-linking of BMP-4 to BRK-3 is undetectable in the absence of either the BRK-1 or BRK-2 BMP type I receptors. Cotransfection of BRK-2 with BRK-3 greatly enhanced affinity labeling of BMP-4 to the type I receptor, in contrast to the affinity labeling pattern observed with the BRK-1 + BRK-3 heteromeric complex. Furthermore, a subpopulation of super-high affinity binding sites is formed in COS cells upon cotransfection only of BRK-2 + BRK-3, suggesting that the different heteromeric BMP receptor complexes have different signaling potential.

Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs.

The mechanism for the rapid desensitization in bradykinin-stimulated inositol monophosphate production in NG108-15 cells involves interaction of a single receptor with multiple signaling pathways.

We have previously shown that the mechanism for the rapid desensitization in bradykinin (BDK)-stimulated inositol monophosphate (IP) production in NG108-15 cells involves both a rapid loss of receptors from the cell surface and an uncoupling of the receptor:G-protein:phospholipase C (PLC) signaling process, with protein kinase C (PKC) activation playing a role only at a postreceptor level (Wolsing and Rosenbaum, 1991). In contrast to BDK, a 5-min pretreatment with the BDK receptor "antagonist" NPC-567 is sufficient to cause a substantial decrease in the subsequent BDK maximal response (Emax) without altering either the BDK potency (EC50) or the BDK receptor number. An 18-hr pretreatment of the cells with 200 ng/ml pertussis toxin (PT) does not alter the BDK response (Fold stim = 2.36 +/- 0.18 vs. 2.00 +/- 0.25 in controls, N = 4), reiterating previous observations that BDK-stimulated IP production in this cell line is mediated by a pertussis toxin (PT)-insensitive G-protein. However, PT pretreatment significantly (P < .05) attenuates the receptor loss that accompanies the desensitization process. Taken together, these data imply that the BDK receptor in NG108-15 cells interacts with both PT-sensitive and PT-insensitive G-proteins. Because NPC-567 pretreatment results in a desensitization that is not accompanied by receptor loss, it appears that NPC-567 is able to facilitate an agonistic interaction with only the PT-insensitive G-proteins that are available to the receptor.

Characterization of the RDC1 gene which encodes the canine homolog of a proposed human VIP receptor. Expression does not correlate with an increase in VIP binding sites.

We have isolated a portion of the canine gene encoding the orphan receptor RDC1 [1]. The complete coding sequence is contained in a single exon, and an intron divides the 5' untranslated region of RDC1 mRNA. The RDC1 protein is 94% homologous to the gene product of GPRN1, which has been proposed to serve as a VIP receptor when expressed in CHO-K1 and COS-7 cells (Sreedharan, S.P. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 4986-4990). Northern analysis indicates that CHO-K1 cells endogenously express a 2.1 kb RDC1 mRNA. However, while CHO-K1 cells possess detectable low affinity [125I]VIP binding sites, VIP binding is not altered in membranes of CHO-K1 cells expressing varying amounts of the RDC1 gene construct. Further, endogenous VIP binding is not increased by transient expression of RDC1 in COS-7 cells. Taken together, the data suggest that RDC1 is not a canine homolog of the proposed VIP receptor.

Bradykinin-stimulated inositol phosphate production in NG108-15 cells is mediated by a small population of binding sites which rapidly desensitize.

[3H]Bradykinin (BDK) binds to two distinct binding sites (P less than .01, N = 12) in NG108-15 cell membranes; (site 1: Kd1 = 3.09 x 10(-10) M, Bmax1 = 242 +/- 24 fmol/mg protein) and (site 2: Kd2 = 1.94 x 10(-8) M, Bmax2 = 491 +/- 75 fmol/mg protein). Although site 1 comprises only 33 +/- 4% (N = 12) of the total binding site population, comparison of the binding affinity and functional potency for BDK agonist analogs exhibiting differential selectivity for the two sites reveals that this high affinity site is the receptor mediating inositol monophosphate (IP) production in this cell line. BDK-stimulated IP production undergoes a very rapid (5 min) desensitization that is characterized by both a loss in agonist potency (EC50 = 3.57 x 10(-9) M vs. 1.94 x 10(-10) M in controls; P less than .001, N = 12) and a decrease in amplitude of response (fold stimulation = 1.45 +/- 0.06 vs. 1.80 +/- 0.09 in controls; P less than .01, N = 12). Only the decrease in response amplitude is attenuated by down-regulation of protein kinase C by prior long term treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), indicating an involvement of protein kinase C activation in the desensitization process. Desensitization is accompanied by down-regulation of site 1 only (Bmax1 = 71 +/- 8 fmol/mg (N = 10; P less than .001 vs. controls)); Bmax2 and the Kd for BDK at both sites remain unchanged, further supporting the contention that site 1 is the functionally relevant receptor. In contrast to the functional data, long term TPA treatment does not attenuate the receptor down-regulation, indicating that the rapid desensitization involves both receptor-related and postreceptor mechanisms. The implications of this property of the BDK receptor for analog design and receptor classification are discussed.