Structure of platelet glycoprotein IIIa. A common subunit for two different membrane receptors.

The platelet membrane glycoprotein IIb/IIIa complex is a member of a family of alpha/beta heterodimers that function as receptors for adhesive proteins. In this report we describe the structure of the human beta subunit GPIIIa deduced from an analysis of 4.0 kb of overlapping cDNA sequences isolated from a human erythroleukemia (HEL) cell cDNA expression library. A continuous open reading frame encoding all 788 amino acids for GPIIIa was present. The deduced amino acid sequence included a 26-residue amino-terminal signal peptide, a 29-residue transmembrane domain near the carboxy terminus, and four tandemly repeated cysteine-rich domains of 33-38 residues. An exact correspondence of 128 amino acids from seven human platelet GPIIIa fragments with HEL GPIIIa indicates that HEL and platelet GPIIIa are the same gene product. The HEL GPIIIa sequence was compared with the sequences of the beta subunit for the human LFA-1/Mac-1/p150.95 complex and human endothelial cell GPIIIa, revealing a 38% similarity with the former and virtual identity with the latter. Northern blot analysis using RNA from both HEL and endothelial cells revealed two GPIIIa transcripts of 5.9 and 4.1 kb. However, HEL RNA, but not endothelial cell RNA, contained a transcript for GPIIb. This indicates that the GPIIIa-containing heterodimers in platelets and endothelial cells are not identical structures, but are members of a subfamily within the human family of adhesion protein receptors sharing an identical beta subunit.

An analysis of polygenes affecting wing shape on chromosome 2 in Drosophila melanogaster.

Genetic effects on an index of wing shape on chromosome 2 of Drosophila melanogaster were mapped using isogenic recombinants with transposable element markers. At least 10 genes with small additive effects are dispersed evenly along the chromosome. Many interactions exist, with only small net effects in homozygous recombinants and little effect on phenotypic variance. Heterozygous chromosome segments show almost no dominance. Pleiotropic effects on leg shape are only minor. At first view, wing shape genes form a rather homogeneous class, but certain complexities remain unresolved.

An analysis of polygenes affecting wing shape on chromosome 3 in Drosophila melanogaster.

Loci on the third chromosome of Drosophila melanogaster that affect an index of wing shape were mapped, using recombinant isogenic lines, with transposable elements as markers. Many genes with small subequal effects are dispersed along the whole chromosome. Their alleles act nearly additively in heterozygotes. They have small correlated effects on leg shape, but no detectable effects on halteres. Small negative net interactions occur over most of the chromosome. The data set of 519 recombinant isogenic lines can be explained reasonably well by two models. One model posits an indefinitely large number of loci with no interactions. The other model posits 11 loci with additive effects whose sum equals the total phenotypic range and with large positive and negative interactions that nearly cancel each other.

Structural and functional comparison of the genes for human platelet factor 4 and PF4alt.

Platelet factor 4 (PF4) is a 70 amino acid heparin-binding protein released from the alpha-granules of activated platelets. Its exact biologic function is not known, although PF4 is a member of a multigene family involved in chemotaxis, coagulation, inflammation, and cell growth. We previously cloned the cDNA for human PF4 from a human erythroleukemic (HEL) cell expression library. We now report the isolation and sequence determination of the gene for human PF4. This gene contains three exons and spans approximately 1,000 basepairs (bp). Concurrently, we have cloned a highly homologous gene that we have called PF4alt. We show that PF4 and PF4alt are non-allelic genes: the human PF4 gene is encoded on a 10 kilobasepairs (kb) EcoRI fragment, and its DNA sequence agrees with protein and cDNA data for PF4, while PF4alt is encoded in a polymorphic 3 or 5 kb EcoRI fragment. Compared with PF4, this gene has 14% DNA and 38% amino acid divergence in the signal peptide region, and 2.6% DNA and 4.3% amino acid divergence in the coding region of the mature protein. PF4alt contains three amino acid substitutions (P58----L, K66----E, and L67----H) near the C-terminus, in a region known to be critical for PF4 function. Primer extension studies show the 5'-untranslated region of PF4 is 73 bp long. A TATA box is present 30 bp 5' to the transcription start site. A 90 bp stretch of pyrimidines (including 53 consecutive thymidine residues) begins at -227 bp and is analogous to a similar region of 30 residues 5' to the rodent PF4 gene. This pyrimidine-rich region is absent from the PF4alt gene; however, DNA homology exists between the two human genes in the 5'- and 3'-flanking regions and extends for over 3.6 kb. Alternating purine/pyrimidine tracts occur both 5' and 3' to PF4 and PF4alt but do not define the endpoints of the gene duplication, which extend beyond these sequences at least at the 5' end. Northern blot analysis using gene-specific oligonucleotides and platelet RNA showed an 800 or 900 nucleotide (n) message for PF4 and PF4alt, respectively. Northern blot and primer extension studies show that steady-state platelet PF4 mRNA levels are approximately one magnitude greater than PF4alt mRNA levels. Thus, these studies demonstrate that PF4alt mRNA is expressed in platelets. Whether PF4alt protein is expressed remains to be determined, and the nature of its biologic function needs to be studied.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors.

The platelet membrane glycoprotein IIb X IIIa heterodimer complex (GPIIb X IIIa) is the platelet receptor for adhesive proteins, containing binding sites for fibrinogen, von Willebrand factor, and fibronectin on activated platelets. GPIIb X IIIa also appears to be a member of a family of membrane adhesive protein receptors that plays a major role in cell-cell and cell-matrix interactions. GPIb is the larger component of this platelet receptor and is composed of two disulfide-linked subunits. In this report we describe the analysis of cDNA clones for human GPIIb that were isolated from a lambda gt11 expression library prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequence, revealing a continuous open reading frame encoding both GPIIb subunits. The cDNA encodes 1039 amino acids: 137 constituting the smaller subunit, 871 constituting the larger subunit, and 30 constituting an NH2-terminal signal peptide. No homology was found between the larger and smaller subunits. The smaller subunit contains a 26-residue hydrophobic sequence near its COOH terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids present in the larger subunit are homologous to the calcium binding sites of calmodulin and troponin C. Northern blot analysis using HEL cell RNA indicated that the mature mRNA coding for GPIIb is 4.1 kilobases in size. A comparison of the GPIIb coding region with available cDNA sequences of the alpha-chains of the vitronectin and fibronectin receptors revealed 41% DNA homology and 74% and 63% amino acid homology, respectively. Our data establish the amino acid sequence for the human platelet glycoprotein IIb and provide additional evidence for the existence of a family of cellular adhesion protein receptors.

Organization of the gene for platelet glycoprotein IIb.

The glycoprotein (GP) IIb/IIIa heterodimer functions as a receptor for fibrinogen, von Willebrand factor, and fibronectin on activated platelets; it is dysfunctional in the bleeding diathesis Glanzmann's thrombasthenia. This receptor is a member of the integrin family, which includes homologous membrane receptors involved in a number of different cell-cell and cell-matrix adhesive interactions. Knowledge of the sequence and organization of the GPIIb and GPIIIa genes will help in understanding evolutionary relationships and functional homologies of this family of adhesion protein receptors and will facilitate analysis of molecular defects responsible for thrombasthenia. Using the GPIIb cDNA as a probe, we have isolated overlapping genomic clones encompassing the entire coding region, the 5'- and 3'-untranslated sequences, and the immediate flanking regions for the GPIIb gene. The gene spans approximately 17.2 kilobases (kb); all but approximately 2.6 kb of intronic DNA sequence has been determined. The GPIIb gene contains 30 exons whose demarcations do not correlate with previously suggested functional domains. Two intron/exon borders have the rare GC splice donor sequence instead of the consensus GT sequence. There are at least seven complete and three partial AluI sequence repeats within the intron sequences. RNase protection, S1 nuclease analysis, and primer extension studies using human erythroleukemia (HEL) cell RNA and platelet RNA map a major transcription start site 32 base pairs (bp) 5' to the beginning of the coding region; however, there are no canonical consensus TATA or CAAT boxes in the region immediately 5' to the proposed cap site. The immediate 5'-flanking sequence of rodent GPIIb demonstrates complete identity near the proposed cap site with its human counterpart, but again, no TATA or CAAT boxes are apparent.

Turnover of the cystic fibrosis transmembrane conductance regulator (CFTR): slow degradation of wild-type and delta F508 CFTR in surface membrane preparations of immortalized airway epithelial cells.

The protein product of the cystic fibrosis (CF) gene, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is known to function as an apical chloride channel at the surface of airway epithelial cells. It has been proposed that CFTR has additional intracellular functions and that there is altered processing of mutant forms. In examining these functions we found a stable form of CFTR with slow turnover in surface membrane preparations from CF and non-CF immortalized airway epithelial cell lines. The methods used to study the turnover of CFTR were pulse/chase experiments utilizing saturation labeling of [35S] Met with chase periods of 5-24 h in the presence of 8 mM Met and cell fractionation techniques. Preparations of morphologically identifiable surface membranes were compared to total cell membrane preparations containing intracellular membranes. Surface membrane CFTR had lower turnover defined by pulse/chase ratios than that of the total cell membrane preparations. Moreover, mutant CFTR was stable in the surface membrane fraction with little degradation even after a 24 h chase, whereas wild-type CFTR had a higher pulse/chase ratio at 24 h. In the presence of 50 microM castanospermine, which is an inhibitor of processing alpha-glucosidases, a more rapid turnover of mutant CFTR was found in the total cell membrane preparation, whereas wild-type CFTR had a lower response. The results are compatible with a pool of CFTR in or near the surface membranes which has an altered turnover in CF and a glycosylation-dependent alteration in the processing of mutant CFTR.