Analysis of Serotonin Transporter in Human Platelets by Immunoblotting Using Site-Specific Antibodies

A. D. Dmitriev^1*, E. V. Pavlova², M. I. Factor², O. L. Segal¹, Yu. S. Massino¹, I. V. Dobrohotov², M. B. Smirnova¹, D. E. Hwun², D. A. Yakovleva², G. I. Kolyaskina², and O. S. Brusov²

¹Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow 117485, Russia; fax: (7-095) 338-8500; E-mail: dmitr@rcmh.msk.ru

²Mental Health Center, Russian Academy of Medical Sciences, Zagorodnoe Shosse 2, Moscow 113152, Russia; fax: (7-095) 952-8940; E-mail: oleg_brusov@hotmail.com

^* To whom correspondence should be addressed.

Received July 29, 2003; Revision received October 31, 2003

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We have produced a panel of site-specific antibodies recognizing different regions of the human serotonin transporter (SERT). This panel included: 1) monoclonal antibodies 23C5 (mAbs 23C5) to the C-terminal region (amino acid residues 597-630); 2) polyclonal antibodies (pAbs) to the N-terminal region (amino acid residues 69-83); 3) pAbs to the region (amino acid residues 86-100) in the beginning of the first transmembrane domain (TMD). The antibodies were produced using recombinant proteins and synthetic peptides (containing certain sequences of SERT) as antigens. These antibodies were purified by affinity chromatography, conjugated to horseradish peroxidase (HRP), and used for immunoblotting analysis of SERT in extracts of human platelets. Sodium dodecyl sulfate extracts were prepared under conditions preventing non-specific proteolytic degradation of the proteins. In platelet extracts, all antibodies were able to detect the 67 kD protein, apparently corresponding to full-length SERT molecule (its theoretical mass is about 70 kD). These antibodies also detected several polypeptides of smaller size (56, 37, 35, 32, 22, and 14 kD), apparently corresponding to N-terminal, C-terminal, and non-terminal SERT fragments. Specificity of immunostaining was confirmed by preincubation of HRP-labeled anti-SERT antibodies with excess of corresponding antigen, which resulted in disappearance of protein band staining. It is suggested that SERT undergoes a programmed proteolytic cleavage (processing) resulting in formation of several SERT-derived polypeptides of smaller size. It is possible that one of the cleaved SERT species is required for serotonin transport activity. Possible sites for specific proteolysis may be located in the region near TMD1 and in the intracellular loop between TMD4 and TMD5.

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KEY WORDS: serotonin transporter, proteolytic processing, human platelets, immunoblotting, site-specific antibodies, monoclonal antibodies

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