Dissociation of the high density lipoprotein and low density lipoprotein binding activities of murine scavenger receptor class B type I (mSR-BI) using retrovirus library-based activity dissection

The murine class B, type I scavenger receptor (mSRBI) is a receptor for bot h high density lipoprotein (HDL) and low density lipoprotein (LDL) and medi ates selective, rather than endocytic, uptake of lipoprotein lipid. We have developed a "retrovirus library-based activity dissection" method to gener ate mSR-BI mutants in which some, but not all, of the activities of this mu ltifunctional protein have been disrupted. This method employs three techni ques: 1) efficient in vitro cDNA mutagenesis there error-prone PCR was used ), 2) efficient retroviral delivery and high expression of single mutant cD NAs into individual cells, and 3) isolation of infected cells expressing th e desired mutant phenotype using high sensitivity positive/negative screeni ng by two-color fluorescence-activated cell sorting. A set of mutants, all having arginine substitutions at two common sites (positions 402 or 401 and position 418), were isolated and characterized. Mutation at either site al one did not generate as strong a mutant phenotype (loss of DiI uptake from DiI-HDL) as did the double mutations. "Activity-dissected" double mutants w ere as effective as wild-type mSR-BI in functioning as LDL receptors, media ting high affinity LDL binding and uptake of metabolically active cholester ol from LDL, but they lost most of their corresponding HDL receptor activit y. Thus, these mutants provide support for the proposal that the interactio n of SR-BI with HDL differs from that with LDL. Examination of the in vivo function of such mutants may provide insights into the differential roles o f the LDL and HDL receptor activities of SR-BI in normal lipoprotein metabo lism and in SR-BI's ability to protect against atherosclerosis.