1912 Volume 4 ‘ Number II 1994 Molecular Characterization of Rat Glomerular Epithelial Cell Complement Receptors1 Richard J. Quigg,2 and Arthur E. Sneed Ill P.J. Quigg, A.E. Sneed Ill, Division of Nephrology, De- partment of Internal Medicine, Medical College of Virginia, Richmond, VA (J. Am. Soc. Nephrol. 1994; 4:1912-1919) ABSTRACT Complement receptor type I (CR1) has previously been isolated from cultured rat glomerular epithelial cells (GEC) by C3b affinity chromatography. In ad- dition, the presence of Crry in GEC and in rat gb- meruli has been demonstrated. Crry appears to be the rodent analogue of human decay accelerating factor, which was previously described in human GEC and in human gbomeruli. In this study, the mo- lecular biology of these rat complement receptors is examined. A specific cDNA probe for rat CR1 was generated by reverse transcription of GEC mRNA, followed by polymerase chain reaction (PCR). The oligonucleotide primers were chosen from con- served regions spanning 27 1 bases in human and mouse CR1. A 271-base-pair PCR product was gen- erated from rat GEC cDNA, the nucleotide sequence of which was 70.1% and 77.2% identical to those of the respective mouse and human sequences. This PCR product, designated rCRI-p, was then used to probe for CR1 mRNA. By northern blot analysis, rCRI- p hybridized to 4.5-kibobase (kb) mRNA from both cultured GEC and rat glomeruli and also weakly hybridized to 4.5-kb CR1 mRNA from mouse spleen. In additional northern blots, a nucleotide probe for mouse Crry hybridized to mRNA of 2. 1 to 2.4 kb from rat GEC, slightly larger than the 1.9- to 2.1-kb mouse Crry mRNA. Therefore, mRNA for CR1 and Crry are present in cultured rat GEC and in rat gbomeruli in vivo. To further investigate the composition of rat CR1 mRNA, northern hybridizations were performed with nucleotide probes for mouse and human CR1. Al- though the nucleotide probe for human CR1 hybrid- , Received September 28. 1993. Accepted February 3. 1994. 2 Correspondence to Dr. R.J. Quigg. Division of Nephrology, Medical College of Virginia. MCV Station, Box 160, Richmond. VA 23298. 1046-6673/0411-1912503.00/0 Journal of the American Society of Nephrology Copyright C 1994 by the American Society of Nephrology ized to rat CR1 mRNA, the probe for mouse CR1 did not. Taken together, the available data indicate that rat CR1 has greater homology to human CR1 than to mouse CR1. Key Words: Glomerulus. complement receptor type 1. Crry. regulators of complement activation, short consensus re- peats S eventeen years ago, receptors for C3 were dem- onstrated on human glomerular epithelial cells (GEC) in vivo (1) and in vitro (2). Once complement receptor type 1 (C3b/C4b receptor; CR 1 ) was purified from human erythrocytes (3) and specific anti-CR 1 antibodies became available, these GEC receptors were documented to be CR1 by immunohistochemi- cab techniques (4). Recently, mRNA for CR! has been localized to human GEC by in situ hybridization (5). In these studies, CR! mRNA was present in fetal gbomerubI but not in adult gbomeruli, despite the abundant presence of CR 1 protein in the batter. The gene for human CR 1 resides in the regulators of complement activation (RCA) gene cluster on chro- mosome 1 (6). CR1 contains the fundamental unit of the RCA family, the short consensus repeat (5CR). Each 5CR is made up of 60 to 70 amino acids and includes a framework of four cysteines (7,8). The most frequently occurring albotype of human CR! contains 30 tandemby arranged 5CR, a 25-amino- acid tnansmembrane domain, and a 43-amino-acid cytoplasmie region. The 28 NH2-terminal 5CR are arranged as four long homologous repeats of 7 SCR each (7). The recent characterization of munine RCA pro- teins has illustrated the evolutionary complexity of this family. The mouse has two proteins of 190 to 2 10 and 60 to 70 kd, designated mouse CR 1 (or Cr2) and Crry (or p65), respectively, that are homologous to human CR! (9-13). Mouse CR1 also has signifi- cant homology to human CR2 and appears to anise via the addition of six CR1-like 5CR onto the N ten- minus of a CR2-like protein (1 3, 1 4). Little is known about RCA proteins in the rat, although mRNA for Crry was recently found in rat neural tissue (15). Earlier studies showed that cultured nat GEC have receptors for C3 fragments on the basis of positive rosetting with complement-coated erythrocytes (1 6, 1 7). By C3b affinity chromatography, we isolated a 200-kd protein from detengent-sobubibized cultured rat GEC that was confirmed to be CR! by immuno-