A common PDGF receptor is activated by homodimeric A and B forms of PDGF

The human platelet-derived growth factor (PDGF) receptor complementary DNA was cloned and expressed by transfection of Chinese hamster ovary (CHO) fibroblasts. The ability of CHO cells expressing the human receptor complementary DNA (CHO-HR5) to interact with different recombinant forms of PDGF (AA and BB homodimers) was tested. Both forms of PDGF bind to the transfected receptor, stimulate the receptor tyrosine kinase activity, and elicit a mitogenic response in a manner that was indistinguishable from the responses of Balb/c 3T3 cells to AA and BB forms of PDGF can be attributed to a single type of receptor and show that the AA form, like the BB form, is a true mitogen.     

Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells

The sequence of the human epidermal growth factor (EGF) receptor shows great homology with the avian erythroblastosis virus v-erb B oncogene, raising the possibility that the receptor gene is identical to the c-erb B protooncogene. Human A431 epidermoid carcinoma cells, which have an unusually high number of EGF receptors, were examined to determine whether elevated EGF receptor levels correlate with gene amplification. Southern blots of genomic DNA's from A431 and other human cell lines were probed with either a v-erb B gene fragment or a human EGF receptor complementary DNA clone (pE7), previously isolated from an A431 complementary DNA library. When either probe was used to analyze Eco RI- or Hind III-generated DNA fragments, EGF receptor DNA sequences were amplified about 30-fold in A431. Differences in the banding pattern of A431 DNA fragments relative to normal fibroblast DNA indicate the occurrence of a rearrangement in the region of the receptor gene. Furthermore, A431 cells contain a characteristic, prominent 2.9-kilobase RNA. These results are consistent with the hypothesis that, in A431 cells, gene amplification, possibly associated with a translocation event, may result in the overproduction of EGF receptor protein or the appearance of the transformed phenotype (or both).     

Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop.

An expression cloning strategy was devised to isolate the keratinocyte growth factor (KGF) receptor complementary DNA. NIH/3T3 fibroblasts, which secrete this epithelial cell-specific mitogen, were transfected with a keratinocyte expression complementary DNA library. Among several transformed foci identified, one demonstrated the acquisition of specific high-affinity KGF binding sites. The pattern of binding competition by related fibroblast growth factors (FGFs) indicated that this receptor had high affinity for acidic FGF as well as KGF. The rescued 4.2-kilobase complementary DNA was shown to encode a predicted membrane-spanning tyrosine kinase related to but distinct from the basic FGF receptor. This expression cloning approach may be generally applicable to the isolation of genes that constitute limiting steps in mitogenic signaling pathways.     

Role of phosphatidylinositol kinase in PDGF receptor signal transduction

The molecules with which the platelet-derived growth factor (PDGF) receptor interacts to elicit the biochemical reactions responsible for cell proliferation have not been identified. Antisera directed against specific PDGF receptor peptides coprecipitated a phosphatidylinositol (PI) kinase and the PDGF receptor. Immunoprecipitates from PDGF-stimulated cells contained 10 to 50 times as much PI kinase as those from unstimulated cells. Mutation of the PDGF receptor by deletion of its kinase insert region resulted in a receptor markedly less effective than the wild type in eliciting cell proliferation and defective in PDGF-stimulated PI kinase, but still capable of PDGF-induced receptor autophosphorylation and phosphoinositide hydrolysis. These data show that the PDGF receptor is physically associated with a PDGF-sensitive PI kinase that is distinct from tyrosine kinase and is not required for PDGF-induced PI hydrolysis. The finding that the mutant PDGF receptor missing the kinase insert domain elicited known early biochemical responses to PDGF, but did not associate with or regulate PI kinase, suggests a novel role for the receptor-associated PI kinase in the transmission of mitogenic signals.     

A large deletion within the T-cell receptor beta-chain gene complex in New Zealand white mice

The T-cell receptor beta-chain gene complex contains a duplication of D beta, J beta, and C beta gene segments in mice and man. When DNA from many inbred strains of mice was screened an unusual allele of the beta locus was identified in New Zealand White (NZW) mice. This allele is distinguished by the deletion of an 8.8-kilobase segment of DNA containing C beta 1, D beta 2 and the J beta 2 cluster. Despite the fact that all NZW T-cell receptors must be derived from a single set of beta-chain gene segments, this strain has functional T cells and is phenotypically normal. This deletion of T-cell receptor beta-chain segments occurs in a strain known to contribute to lupus-like autoimmune disease.     

Cloning of human androgen receptor complementary DNA and localization to the X chromosome

The androgen receptor (AR) mediates the actions of male sex steroids. Human AR genomic DNA was cloned from a flow-sorted human X chromosome library by using a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors. The AR gene was localized on the human X chromosome between the centromere and q13. Cloned complementary DNA, selected with an AR-specific oligonucleotide probe, was expressed in monkey kidney (COS) cells and yielded a high-affinity androgen-binding protein with steroid-binding specificity corresponding to that of native AR. A predominant messenger RNA species of 9.6 kilobases was identified in human, rat, and mouse tissues known to contain AR and was undetectable in tissues lacking AR androgen-binding activity, including kidney and liver from androgen-insensitive mice. The deduced amino acid sequence of AR within the DNA-binding domain has highest sequence identity with the progesterone receptor.     

Human CSF-1: molecular cloning and expression of 4-kb cDNA encoding the human urinary protein

A 4-kilobase complementary DNA (cDNA) encoding human macrophage-specific colony-stimulating factor (CSF-1) was isolated. When introduced into mammalian cells, this cDNA directs the expression of CSF-1 that is structurally and functionally indistinguishable from the natural human urinary CSF-1. Direct structural analysis of both the recombinant CSF-1 and the purified human urinary protein revealed that these species contain a sequence of at least 40 amino acids at their carboxyl termini which are not found in the coding region of a 1.6-kilobase CSF-1 cDNA that was previously described. These results demonstrate that the human CSF-1 gene can be expressed to yield at least two different messenger RNA species that encode distinct but related forms of CSF-1.     

Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family

A complementary DNA (cDNA) for the rat luteal lutropin-choriogonadotropin receptor (LH-CG-R) was isolated with the use of a DNA probe generated in a polymerase chain reaction with oligonucleotide primers based on peptide sequences of purified receptor protein. As would be predicted from the cDNA sequence, the LH-CG-R consists of a 26-residue signal peptide, a 341-residue extracellular domain displaying an internal repeat structure characteristic of members of the leucine-rich glycoprotein (LRG) family, and a 333-residue region containing seven transmembrane segments. This membrane-spanning region displays sequence similarity with all members of the G protein-coupled receptor family. Hence, the LH-CG-R gene may have evolved by recombination of LRG and G protein-coupled receptor genes. Cells engineered to express LH-CG-R cDNA bind human choriogonadotropin with high affinity and show an increase in cyclic adenosine monophosphate when exposed to hormone. As revealed by RNA blot analysis and in situ hybridization, the 4.4-kilobase cognate messenger RNA is prominently localized in the rat ovary.     

Two classes of PDGF receptor recognize different isoforms of PDGF

Previous studies involving platelet-derived growth factor (PDGF) have been based on the premise that a single cell-surface receptor binds all three isoforms of PDGF (AA, BB, and AB). It is now shown that two populations of PDGF receptor exist and can be distinguished by their ligand binding specificity. The B receptor binds only the BB dimer, whereas the A/B receptor binds AA, BB, and AB dimers. Human dermal fibroblasts appear to express seven times as much B receptor as A/B receptor. The B receptor is responsible for most PDGF receptor phosphorylation.     

Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) participates in many processes including early developmental events, angiogenesis, wound healing, and maintenance of neuronal cell viability. A 130-kilodalton protein was isolated on the basis of its ability to specifically bind to bFGF. A complementary DNA clone was isolated with an oligonucleotide probe corresponding to determined amino acid sequences of tryptic peptide fragments of the purified protein. The putative bFGF receptor encoded by this complementary DNA is a transmembrane protein that contains three extracellular immunoglobulin-like domains, an unusual acidic region, and an intracellular tyrosine kinase domain. These domains are arranged in a pattern that is different from that of any growth factor receptor described.     

Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization

Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.     

Sequence and expression of human estrogen receptor complementary DNA

The mechanism by which the estrogen receptor and other steroid hormone receptors regulate gene expression in eukaryotic cells is not well understood. In this study, a complementary DNA clone containing the entire translated portion of the messenger RNA for the estrogen receptor from MCF-7 human breast cancer cells was sequenced and then expressed in Chinese hamster ovary (CHO-K1) cells to give a functional protein. An open reading frame of 1785 nucleotides in the complementary DNA corresponded to a polypeptide of 595 amino acids and a molecular weight of 66,200, which is in good agreement with published molecular weight values of 65,000 to 70,000 for the estrogen receptor. Homogenates of transformed Chinese hamster ovary cells containing a protein that bound [3H]estradiol and sedimented as a 4S complex in salt-containing sucrose gradients and as an 8 to 9S complex in the absence of salt. Interaction of this receptor-[3H]estradiol complex with a monoclonal antibody that is specific for primate ER confirms the identity of the expressed complementary DNA as human estrogen receptor. Amino acid sequence comparisons revealed significant regional homology among the human estrogen receptor, the human glucocorticoid receptor, and the putative v-erbA oncogene product. This suggests that steroid receptor genes and the avian erythroblastosis viral oncogene are derived from a common primordial gene. The homologous region, which is rich in cysteine, lysine, and arginine, may represent the DNA-binding domain of these proteins.     

Evidence that the v-sis gene product transforms by interaction with the receptor for platelet-derived growth factor

A scheme for partial purification of biologically active v-sis-coded protein from cells transformed with simian sarcoma virus (SSV) has made possible a functional comparison of the transforming protein with platelet-derived growth factor (PDGF). The SSV-transforming gene product is capable of specifically binding PDGF receptors, stimulating tyrosine phosphorylation of PDGF receptors, and inducing DNA synthesis in quiescent fibroblasts. Each of these activities was specifically inhibited by antibodies to different regions of the v-sis gene product. Moreover, viral infection of a variety of cell types revealed a strict correlation between those cells possessing PDGF receptors and those susceptible to transformation by SSV. These findings provide evidence that SSV-transforming activity is mediated by the interaction of a virus-coded mitogen with PDGF receptors.     

Localization of the gene encoding the human interleukin-2 receptor on chromosome 10

The human interleukin-2 receptor is an inducible growth factor receptor present on the surface of activated T lymphocytes. The receptor is required for a normal T-cell immune response. High-resolution fluorescence-activated chromosome sorting and DNA spot-blot analysis with complementary DNA's for the interleukin-2 receptor indicated that the receptor gene was located on chromosome 9, 10, 11, or 12. In situ hybridization studies showed that the interleukin-2 receptor gene is on the short arm of chromosome 10, p14----15.     

Gene for alpha-chain of human T-cell receptor: location on chromosome 14 region involved in T-cell neoplasms

A human complementary DNA clone specific for the alpha-chain of the T-cell receptor and a panel of rodent X human somatic cell hybrids were used to map the alpha-chain gene to human chromosome 14 in a region proximal to the immunoglobulin heavy chain locus. Analysis by means of in situ hybridization of human metaphase chromosomes served to further localize the alpha-chain gene to region 14q11q12, which is consistently involved in translocations and inversions detectable in human T-cell leukemias and lymphomas. Thus, the locus for the alpha-chain T-cell receptor may participate in oncogene activation in T-cell tumors.     

Beta-adrenergic receptor kinase: primary structure delineates a multigene family

The beta-adrenergic receptor kinase (beta-ARK), which specifically phosphorylates only the agonist-occupied form of the beta-adrenergic and closely related receptors, appears to be important in mediating rapid agonist-specific (homologous) desensitization. The structure of this enzyme was elucidated by isolating clones from a bovine brain complementary DNA library through the use of oligonucleotide probes derived from partial amino acid sequence. The beta-ARK cDNA codes for a protein of 689 amino acids (79.7 kilodaltons) with a protein kinase catalytic domain that bears greatest sequence similarity to protein kinase C and the cyclic adenosine monophosphate (cyclic AMP)--dependent protein kinase. When this clone was inserted into a mammalian expression vector and transfected into COS-7 cells, a protein that specifically phosphorylated the agonist-occupied form of the beta 2-adrenergic receptor and phosphorylated, much more weakly, the light-bleached form of rhodopsin was expressed. RNA blot analysis revealed a messenger RNA of four kilobases with highest amounts in brain and spleen. Genomic DNA blot analysis also suggests that beta-ARK may be the first sequenced member of a multigene family of receptor kinases.