CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumor growth1-3. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signaling pathways result in neutrophil migration to the site of inflammation2. CXCR1 is a class-A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors4-7. Despite its importance, its molecular mechanism is poorly understood due to the limited structural information available. Recently, structure determination of GPCRs has advanced by tailoring the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences8, as well as addition of stabilizing antibodies and small molecules9 that facilitate crystallization in cubic phase monoolein mixtures10. The intracellular loops of GPCRs are critical for G-protein interactions11 and activation of CXCR1 involves both N-terminal residues and extracellular loops2,12,13. Our previous NMR studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity14. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed.

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We have used polyclonal anti-synthetic peptide serum to study the role of retinoblastoma gene (RB) inactivation in a variety of human tumor cell lines. Our analysis indicates that inactivation of the RB protein, p105-Rb, is universal in retinoblastoma cells, vindicating the predictions of the Knudson "two-hit" hypothesis. In addition, our analysis has shown that inactivations of the RB gene are nearly as frequent in a more common human tumor, small cell lung carcinoma. One-third of bladder carcinomas surveyed also carry altered or absent p105-Rb. Other human tumors by contrast demonstrate only infrequent inactivation of the RB gene. These results suggest that inactivation of the RB gene is a critical step in the pathogenesis of a subset of human tumors.The recent isolation of molecular clones of the retinoblastoma gene (RB) has made it possible to evaluate the role of RB gene inactivation in the genesis of a variety of human tumors (1-3). These molecular analyses have confirmed and extended earlier karyotypic and genetic studies that had implied that both copies ofthis gene suffer inactivation during the formation of retinoblastomas and several types of sarcoma (4-10). Since the inactivation ofRB function appears to trigger tumorigenesis, the RB gene has been termed a "tumor suppressor" gene, whose expression is required to constrain normal cellular proliferation.Analysis of the protein product encoded by the RB gene, p105-Rb, has shown it to be a nuclear phosphoprotein having an affinity for DNA (11,12 . This complex formation is apparently central to the ability of these oncoproteins to transform primary cells (14,17).In the initial studies designed to detect inactivation of chromosomal copies of the RB gene, cloned segments of the RB gene were used as probes in Southern blot analysis of tumor DNAs (1)(2)(3)(18)(19)(20)(21) MATERIALS AND METHODSPreparation of Cell Lysates and Immunoprecipitation of the RB Protein from Human Tumor Cells. Tumor cell cultures were incubated for 3-5 hr with [35S~methionine, and cell lysates were prepared as described (13). Immunoprecipitations were performed with rabbit serum no. 147, 144, or 140 (13), and precipitates were resolved by electrophoresis for 15 hr on SDS/polyacrylamide gels, processed for fluorography, and exposed for 1-3 days at -70'C. Bladder, colon, and breast carcinoma cultures were acquired from the American Type Culture Collection as were two retinoblastoma cultures (Y79 and WERI-1). The derivation ofall other retinoblastoma cultures and small cell lung carcinoma (SCLC) cultures has been described (1,23,24). Melanoma tumor cells were a kind gift of Nick Dracopoli of the Massachusetts Institute of Technology.Amplification ofTumor Cell mRNA by Using the Polymerase Chain Reaction (PCR) Technique and DNA Sequence Analysis. RB mRNA was specifically amplified by using oligonucleotide primers from the RB cDNA sequence and the PCR as described (12). Amplified fragments were cloned into M13 and sequenced on both strands by the dideoxy chain termination techniq...

High-resolution solid-state NMR spectra can be obtained from uniformly (15)N-labeled membrane proteins in magnetically aligned bicelles. Fast uniaxial diffusion about the axis of the bilayer normal results in single-line spectra that contain the orientational information necessary for protein structure determination.

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